A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

PubMed Central

Suzuki, Yasuhiro; Kagawa, Naoko; Fujino, Toru; Sumiya, Tsuyoshi; Andoh, Taichi; Ishikawa, Kumiko; Kimura, Rie; Kemmochi, Kiyokazu; Ohta, Tsutomu; Tanaka, Shigeo

2005-01-01

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. PMID:16009811

Enhanced detection of fluorescence quenching in labeled cells

DOEpatents

Crissman, Harry A.; Steinkamp, John A.

1992-01-01

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence that is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is substracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle.

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

Evaluation of trauma care using TRISS method: the role of adjusted misclassification rate and adjusted w-statistic.

PubMed

Llullaku, Sadik S; Hyseni, Nexhmi Sh; Bytyçi, Cen I; Rexhepi, Sylejman K

2009-01-15

Major trauma is a leading cause of death worldwide. Evaluation of trauma care using Trauma Injury and Injury Severity Score (TRISS) method is focused in trauma outcome (deaths and survivors). For testing TRISS method TRISS misclassification rate is used. Calculating w-statistic, as a difference between observed and TRISS expected survivors, we compare our trauma care results with the TRISS standard. The aim of this study is to analyze interaction between misclassification rate and w-statistic and to adjust these parameters to be closer to the truth. Analysis of components of TRISS misclassification rate and w-statistic and actual trauma outcome. The component of false negative (FN) (by TRISS method unexpected deaths) has two parts: preventable (Pd) and non-preventable (nonPd) trauma deaths. Pd represents inappropriate trauma care of an institution; otherwise nonpreventable trauma deaths represents errors in TRISS method. Removing patients with preventable trauma deaths we get an Adjusted misclassification rate: (FP + FN - Pd)/N or (b+c-Pd)/N. Substracting nonPd from FN value in w-statistic formula we get an Adjusted w-statistic: [FP-(FN - nonPd)]/N, respectively (FP-Pd)/N, or (b-Pd)/N). Because adjusted formulas clean method from inappropriate trauma care, and clean trauma care from the methods error, TRISS adjusted misclassification rate and adjusted w-statistic gives more realistic results and may be used in researches of trauma outcome.

Synthesis and Electrophoretic Properties of Novel Nanoparticles for Colored Electronic Ink and e-Paper Applications.

PubMed

Jablonski, Christelle; Grundler, Gerhard; Pieles, Uwe; Stebler, Simon; Oehrlein, Reinhold; Szamel, Zbigniew

2016-01-01

A new approach based on non-pigmented, stable colored nanoparticles able to migrate upon application of an electrical field (10-60 V) has been developed for the improvement of the color brightness of e-displays. The scientific challenges comprised the development of efficient syntheses of tri- and bifunctional dendrimers including branching points for further extension and individual decoration with dye (yellow, magenta, cyan). The covalent attachment of these scaffolds to silica nanoparticles was performed via hydrosilylation and final in situ charging generated attractive silica shells for the substractive CMY color space model.

Sustained/Continuous Operations Subgroup of the Department of Defense Human Factors Engineering Technical Group: Program Summary and Substracts from the Semiannual Meeting (9th) Held in Pensacola, Florida on 11-12 July 1989.

DTIC Science & Technology

1990-03-01

User’s Guide will include the guidelines on the use of pharmacological agents in the context of sleep management . The nature of short sleep (napping) is...are used to evaluate effects and the mechanisms of action of pharmacological agents. Results and Future Obiectives Benzodiazepines and tryptophan...Arlington, ViA 22202.4302. arid to the Offi(e of Management and Budget. Paperwork RedLction Project (0704.0188), Washington, DC 20503. 1. AGENCY USE

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

PubMed

Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

2012-01-01

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, C.M.; Berg, D.E.; Wang, G.

1997-07-08

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed. 4 figs.

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, Claire M.; Berg, Douglas E.; Wang, Gan

1997-01-01

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed.

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

PubMed Central

Chen, Yunjia; Qiu, Shihong; Luan, Chi-Hao; Luo, Ming

2007-01-01

Background Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics. Results With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies. Conclusion The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics. PMID:17663785

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Physical mapping of complex genomes

DOEpatents

Evans, G.A.

1993-06-15

A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.

The influence of alternative plant propagation and stand establishment techniques on survival and growth of eastern cottonwood (Populus deltoids Bartr.) clones

Treesearch

Donald J. Kaczmarek; Randall Rousseau; Jeff A. Wright; Brian Wachelka

2014-01-01

Four eastern cottonwood clones, including standard operational clone ST66 and three advanced clonal selections were produced and included in a test utilizing five different plant propagation methods. Despite relatively large first-year growth differences among clones, all clones demonstrated similar responses to the treatments and clone × cutting treatment interactions...

Optimal quantum cloning based on the maximin principle by using a priori information

NASA Astrophysics Data System (ADS)

Kang, Peng; Dai, Hong-Yi; Wei, Jia-Hua; Zhang, Ming

2016-10-01

We propose an optimal 1 →2 quantum cloning method based on the maximin principle by making full use of a priori information of amplitude and phase about the general cloned qubit input set, which is a simply connected region enclosed by a "longitude-latitude grid" on the Bloch sphere. Theoretically, the fidelity of the optimal quantum cloning machine derived from this method is the largest in terms of the maximin principle compared with that of any other machine. The problem solving is an optimization process that involves six unknown complex variables, six vectors in an uncertain-dimensional complex vector space, and four equality constraints. Moreover, by restricting the structure of the quantum cloning machine, the optimization problem is simplified as a three-real-parameter suboptimization problem with only one equality constraint. We obtain the explicit formula for a suboptimal quantum cloning machine. Additionally, the fidelity of our suboptimal quantum cloning machine is higher than or at least equal to that of universal quantum cloning machines and phase-covariant quantum cloning machines. It is also underlined that the suboptimal cloning machine outperforms the "belt quantum cloning machine" for some cases.

Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

PubMed

Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

PubMed Central

Fu, Changlin; Donovan, William P.; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H.

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%. PMID:25551825

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang Baolong; Department of Mathematics and Physics, Hefei University, Hefei, 230022; Song Qingming

We present a scheme to realize a special quantum cloning machine in separate cavities. The quantum cloning machine can copy the quantum information from a photon pulse to two distant atoms. Choosing the different parameters, the method can perform optimal symmetric (asymmetric) universal quantum cloning and optimal symmetric (asymmetric) phase-covariant cloning.

Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

PubMed

Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

2011-03-01

As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment. Copyright © 2011 Elsevier Inc. All rights reserved.

Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints.

PubMed

Ulrich, Alexander; Andersen, Kasper R; Schwartz, Thomas U

2012-01-01

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

Physical mapping of complex genomes

DOEpatents

Evans, Glen A.

1993-01-01

Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.

To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

PubMed Central

Winters, Misa; Barta, Jodi Lynn; Monroe, Cara; Kemp, Brian M.

2011-01-01

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous. PMID:21738625

Comparative study of biological and technological characters in three generations of silkworm Bombyx mori L. ameiotic, parthenogenetically cloned lines.

PubMed

Greiss, H; Vassilieva, J; Petkov, N; Petkov, Z

2004-11-01

Detect any deviation in biologic and technologic characters of eight ameiotic-parthenogenetically cloned lines of Bombyx mori L. from different origins from a normal sexually reproduced control line in three generations. Comparative study of the three generations was conducted in SES, Vratza, unit of the National Center for Agrarian Sciences of Bulgaria after fixing all environmental rearing conditions. The ameiotic-parthen-clones displayed good parthenogenetic development, although total hatchability was significantly less than the sexually reproducing control populations. Survival rates between clones and control were not significantly different. All clones displayed significantly longer larval periods. Slight decline in second generation, and a steeper one in the third generation were observed for all eight cloned lines in cocoon weight, shell weight, and shell ratio and these differences were statistically significant. Cocoon yield was significantly lower than the control throughout the three generations. Our parthen-cloning method has a high rate of success in comparison to other cloning methods, the cloned progeny populations although were weaker technologically (cocoon weight, shell weight, and shell ratio), the biological characters (parthenogenetic development and survival rate) were not compromised. Further study is needed to determine the thermal needs of the cloned embryos and metabolic rate of all stages.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

PubMed

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Production of cloned calves using roscovitine-treated adult somatic cells as donors.

PubMed

Miyoshi, Kazuchika; Arat, Sezen; Stice, Steven L

2006-01-01

The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize adult bovine granulosa cells in the G0/G1 cell cycle stage. The resulting fetal and calf survival after transfer of cloned embryos was enhanced in the roscovitine-treated group compared with serum-starved controls. The methods described in this chapter outline (1) the preparation of donor cells, (2) the preparation of recipient oocytes, and (3) the production of cloned embryos. The first section involves methods for the preparation of donor cell stocks from isolated granulosa cells and the roscovitine treatment of the cells before nuclear transfer. The second section explains procedures of in vitro maturation of recipient oocytes. The last section involves methods for the production of cell-oocyte complexes, the fusion of the complexes, and the activation, in vitro culture, and transfer into recipient females of cloned embryos.

A rapid and cost-effective method for sequencing pooled cDNA clones by using a combination of transposon insertion and Gateway technology.

PubMed

Morozumi, Takeya; Toki, Daisuke; Eguchi-Ogawa, Tomoko; Uenishi, Hirohide

2011-09-01

Large-scale cDNA-sequencing projects require an efficient strategy for mass sequencing. Here we describe a method for sequencing pooled cDNA clones using a combination of transposon insertion and Gateway technology. Our method reduces the number of shotgun clones that are unsuitable for reconstruction of cDNA sequences, and has the advantage of reducing the total costs of the sequencing project.

Surpassing the no-cloning limit with a heralded hybrid linear amplifier for coherent states

PubMed Central

Haw, Jing Yan; Zhao, Jie; Dias, Josephine; Assad, Syed M.; Bradshaw, Mark; Blandino, Rémi; Symul, Thomas; Ralph, Timothy C.; Lam, Ping Koy

2016-01-01

The no-cloning theorem states that an unknown quantum state cannot be cloned exactly and deterministically due to the linearity of quantum mechanics. Associated with this theorem is the quantitative no-cloning limit that sets an upper bound to the quality of the generated clones. However, this limit can be circumvented by abandoning determinism and using probabilistic methods. Here, we report an experimental demonstration of probabilistic cloning of arbitrary coherent states that clearly surpasses the no-cloning limit. Our scheme is based on a hybrid linear amplifier that combines an ideal deterministic linear amplifier with a heralded measurement-based noiseless amplifier. We demonstrate the production of up to five clones with the fidelity of each clone clearly exceeding the corresponding no-cloning limit. Moreover, since successful cloning events are heralded, our scheme has the potential to be adopted in quantum repeater, teleportation and computing applications. PMID:27782135

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deymier, Martin J., E-mail: mdeymie@emory.edu; Claiborne, Daniel T., E-mail: dclaibo@emory.edu; Ende, Zachary, E-mail: zende@emory.edu

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmittedmore » genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.« less

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

PubMed

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

PubMed

Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

2014-01-01

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

PubMed Central

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2014-01-01

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. PMID:20085739

2p2h effects on the weak pion production cross section

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mariano, A.; Barbero, C.

2015-05-15

The ν{sub l}n → l{sup −}p QE reaction on the A-target is used as a signal event or/and to reconstruct the neutrino energy, using two-body kinematics. Competition of another processes could lead to misidentification of the arriving neutrinos, being important the fake events coming from the CC1π background. A precise knowledge of cross sections is a prerequisite in order to make simulations in event generators to substract the fake ones from the QE countings, and in this contribution we analyze the different nuclear effects on the CC1π channel. Our calculations also can be extended for the NC case.

Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome.

PubMed Central

Knott, V; Rees, D J; Cheng, Z; Brownlee, G G

1988-01-01

Sets of overlapping cosmid clones generated by random sampling and fingerprinting methods complement data at pyrB (96.5') and oriC (84') in the published physical map of E. coli. A new cloning strategy using sheared DNA, and a low copy, inducible cosmid vector were used in order to reduce bias in libraries, in conjunction with micro-methods for preparing cosmid DNA from a large number of clones. Our results are relevant to the design of the best approach to the physical mapping of large genomes. PMID:2834694

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

PubMed Central

Ulrich, Alexander; Andersen, Kasper R.; Schwartz, Thomas U.

2012-01-01

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts. PMID:23300917

Importance sampling with imperfect cloning for the computation of generalized Lyapunov exponents

NASA Astrophysics Data System (ADS)

Anteneodo, Celia; Camargo, Sabrina; Vallejos, Raúl O.

2017-12-01

We revisit the numerical calculation of generalized Lyapunov exponents, L (q ) , in deterministic dynamical systems. The standard method consists of adding noise to the dynamics in order to use importance sampling algorithms. Then L (q ) is obtained by taking the limit noise-amplitude → 0 after the calculation. We focus on a particular method that involves periodic cloning and pruning of a set of trajectories. However, instead of considering a noisy dynamics, we implement an imperfect (noisy) cloning. This alternative method is compared with the standard one and, when possible, with analytical results. As a workbench we use the asymmetric tent map, the standard map, and a system of coupled symplectic maps. The general conclusion of this study is that the imperfect-cloning method performs as well as the standard one, with the advantage of preserving the deterministic dynamics.

Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

PubMed

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-08

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. Copyright 2010 Elsevier Inc. All rights reserved.

Productivity of Populus in monoclonal and polyclonal blocks at three spacings.

Treesearch

Dean S. DeBell; Constance A. Harrington

1997-01-01

Four Populus clones were grown at three spacings (0.5, 1.0, and 1.5 m) in monoclonal plots and in polyclonal plots with all clones in intimate mixture. After the third year, many individual tree and stand traits differed significantly by clone, spacing, deployment method, and their interactions. Differences among clones in growth and stem form were...

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Analysing malaria drug trials on a per-individual or per-clone basis: a comparison of methods.

PubMed

Jaki, Thomas; Parry, Alice; Winter, Katherine; Hastings, Ian

2013-07-30

There are a variety of methods used to estimate the effectiveness of antimalarial drugs in clinical trials, invariably on a per-person basis. A person, however, may have more than one malaria infection present at the time of treatment. We evaluate currently used methods for analysing malaria trials on a per-individual basis and introduce a novel method to estimate the cure rate on a per-infection (clone) basis. We used simulated and real data to highlight the differences of the various methods. We give special attention to classifying outcomes as cured, recrudescent (infections that never fully cleared) or ambiguous on the basis of genetic markers at three loci. To estimate cure rates on a per-clone basis, we used the genetic information within an individual before treatment to determine the number of clones present. We used the genetic information obtained at the time of treatment failure to classify clones as recrudescence or new infections. On the per-individual level, we find that the most accurate methods of classification label an individual as newly infected if all alleles are different at the beginning and at the time of failure and as a recrudescence if all or some alleles were the same. The most appropriate analysis method is survival analysis or alternatively for complete data/per-protocol analysis a proportion estimate that treats new infections as successes. We show that the analysis of drug effectiveness on a per-clone basis estimates the cure rate accurately and allows more detailed evaluation of the performance of the treatment. Copyright © 2012 John Wiley & Sons, Ltd.

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

PubMed Central

Kouprina, Natalay; Larionov, Vladimir

2016-01-01

Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology. PMID:27116033

Cloning of pigs from somatic cells and its prospects.

PubMed

Onishi, Akira

2002-01-01

The technology of somatic cell cloning in pigs is valuable for agricultural and therapeutic purposes. This paper will focus on the current methods of cloning pigs, including our successful microinjection#10; of somatic cell nuclei and its application. #10;

The High Diversity of MRSA Clones Detected in a University Hospital in Istanbul

PubMed Central

Oksuz, Lutfiye; Dupieux, Celine; Tristan, Anne; Bes, Michele; Etienne, Jerome; Gurler, Nezahat

2013-01-01

Background: To characterize the methicillin-resistant Staphylococcus aureus (MRSA) clones present in Istanbul, 102 MRSA isolates collected during a 5-year period at the Istanbul Medical Faculty Hospital were characterized using microarray analysis and phenotypic resistance profiles. Methods: Resistance to methicillin was detected with a cefoxitin disk diffusion assay and confirmed with a MRSA-agar and MRSA detection kit. Antimicrobial susceptibility testing was performed by a disk diffusion assay and interpreted according to the 2012 guidelines of the Antibiogram Committee of the French Society for Microbiology. Decreased susceptibility to glycopeptides was confirmed using the population analysis profile-area under the curve (PAP-AUC) method. The presence of the mecA gene was detected by polymerase chain reaction. Bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits. Strains were extensively characterized using the DNA microarray. Results: Isolates were grouped into six clonal complexes. The most frequently detected clone was the Vienna/Hungarian/Brazilian clone (ST239-MRSA-III), which accounted for 53.9% of the isolates. These isolates were resistant to multiple antibiotics, particularly penicillin, tetracycline, rifampicin, kanamycin, tobramycin, gentamicin, levofloxacin, erythromycin, lincomycin and fosfomycin. Furthermore, three isolates were detected by population analysis profile as heterogeneous vancomycin-intermediate S. aureus (hVISA). The UK-EMRSA-15 clone (ST22-MRSA-IV PVL negative) was detected in 9.8% of the isolates and was mainly susceptible to all anti-staphylococcal antibiotics. Seven isolates (6.9%) were positive for PVL genes and were assigned to the CC80-MRSA-IV clone (European CA-MRSA clone, three isolates), ST8-MRSA-IV clone (USA300 clone, two isolates, one ACME-positive) or ST22-MRSA-IV clone (“Regensburg EMRSA” clone, two isolates). All other clones were detected in one to six isolates and corresponded to well-known clones (e.g., Pediatric clone, Dublin EMRSA clone, WA MRSA-54/63, WA MRSA-1/57). Conclusions: This work highlighted both the high prevalence of ST239-MRSA-III clone and the large diversity of the other MRSA clones detected in a university hospital in Istanbul. PMID:24151444

To clone or not to clone--a Jewish perspective.

PubMed Central

Lipschutz, J H

1999-01-01

Many new reproductive methods such as artificial insemination, in vitro fertilisation, freezing of human embryos, and surrogate motherhood were at first widely condemned but are now seen in Western society as not just ethically and morally acceptable, but beneficial in that they allow otherwise infertile couples to have children. The idea of human cloning was also quickly condemned but debate is now emerging. This article examines cloning from a Jewish perspective and finds evidence to support the view that there is nothing inherently wrong with the idea of human cloning. A hypothesis is also advanced suggesting that even if a body was cloned, the brain, which is the essence of humanity, would remain unique. This author suggests that the debate should be changed from "Is cloning wrong?" to "When is cloning wrong?". PMID:10226913

PyClone: statistical inference of clonal population structure in cancer.

PubMed

Roth, Andrew; Khattra, Jaswinder; Yap, Damian; Wan, Adrian; Laks, Emma; Biele, Justina; Ha, Gavin; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

2014-04-01

We introduce PyClone, a statistical model for inference of clonal population structures in cancers. PyClone is a Bayesian clustering method for grouping sets of deeply sequenced somatic mutations into putative clonal clusters while estimating their cellular prevalences and accounting for allelic imbalances introduced by segmental copy-number changes and normal-cell contamination. Single-cell sequencing validation demonstrates PyClone's accuracy.

Genotype-environment interaction and stability in ten-year height growth of Norway spruce Clones (Picea abies Karst.).

Treesearch

J.B. St. Clair; J. Kleinschmit

1986-01-01

Norway spruce cuttings of 40 clones were tested on seven contrasting sites in northern Germany. Analysis of variance for ten-year height growth indicate a highly significant clone x site interaction. This interaction may be reduced by selection of stable clones. Several measures of stability were calculated and discussed. Characterization of sites by the method of...

Marshall Barber and the century of microinjection: from cloning of bacteria to cloning of everything.

PubMed

Korzh, Vladimir; Strähle, Uwe

2002-08-01

A hundred years ago, Dr. Marshall A. Barber proposed a new technique - the microinjection technique. He developed this method initially to clone bacteria and to confirm the germ theory of Koch and Pasteur. Later on, he refined his approach and was able to manipulate nuclei in protozoa and to implant bacteria into plant cells. Continuous improvement and adaptation of this method to new applications dramatically changed experimental embryology and cytology and led to the formation of several new scientific disciplines including animal cloning as one of its latest applications. Interestingly, microinjection originated as a method at the crossroad of bacteriology and plant biology, demonstrating once again the unforeseen impact that basic research in an unrelated field can have on the development of entirely different disciplines.

Genetic characterization and barcoding of taxa in the genus Wolffia Horkel ex Schleid. (Lemnaceae) as revealed by two plastidic markers and amplified fragment length polymorphism (AFLP).

PubMed

Bog, Manuela; Schneider, Philipp; Hellwig, Frank; Sachse, Svea; Kochieva, Elena Z; Martyrosian, Elena; Landolt, Elias; Appenroth, Klaus-J

2013-01-01

The genus Wolffia of the duckweed family (Lemnaceae) contains the smallest flowering plants. Presently, 11 species are recognized and categorized mainly on the basis of morphology. Because of extreme reduction of structure of all species, molecular methods are especially required for barcoding and identification of species and clones of this genus. We applied AFLP combined with Bayesian analysis of population structure to 66 clones covering all 11 species. Nine clusters were identified: (1) W. angusta and W. microscopica (only one clone), (2) W. arrhiza, (3) W. cylindracea (except one clone that might be a transition form), (4) W. australiana, (5) W. globosa, (6) W. globosa, W. neglecta, and W. borealis, (7) W. brasiliensis, and W. columbiana, (8) W. columbiana, (9) W. elongata. Furthermore, we investigated the sequences of plastidic regions rps16 (54 clones) and rpl16 (55 clones), and identified the following species: W. angusta, W. australiana, W. brasiliensis, W. cylindracea, W. elongata, W. microscopica, and W. neglecta. Wolffia globosa has been separated into two groups by both methods. One group which consists only of clones from North America and East Asia was labelled here "typical W. globosa". The other group of W. globosa, termed operationally "W. neglecta", contains also clones of W. neglecta and shows high similarity to W. borealis. None of the methods recognized W. borealis as a distinct species. Although each clone could be characterized individually by AFLP and plastidic sequences, and most species could be bar-coded, the presently available data are not sufficient to identify all taxa of Wolffia.

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

PubMed Central

Meghdadi, Hossein; Khosravi, Azar D.; Ghadiri, Ata A.; Sina, Amir H.; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15–100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results. PMID:26191059

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

PubMed

Meghdadi, Hossein; Khosravi, Azar D; Ghadiri, Ata A; Sina, Amir H; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15-100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

PubMed Central

Rodríguez, Lleretny; Navarrete, Felipe I.; Tovar, Heribelt; Cox, José F.

2008-01-01

Purpose In order to simplify cloning, a new method that does not require micromanipulators was used. We aimed to evaluate the developmental potential of two bovine cell lines upon cloning. Materials and methods In vitro matured bovine oocytes, were released from zona pellucida, enucleated, fused to foetal or adult somatic donor cells. The reconstructed embryos were reprogrammed, activated and cultured until blastocyst stage. No micromanipulators were used. Blastocyst rate and quality was scored. Some expanded (d7) blastocysts were transferred to recipient cattle and collected back at d17 to assess elongation. Results High developmental potential in vitro of cloned embryos to expanded (d7) blastocysts was achieved (52.6%). In one cell line, 65.7% of blastocysts was scored. Most blastocysts (87.4%) were graded as excellent. In vivo development to elongation (day-17) in temporary recipient cows also showed a high developmental potential (11/18 transferred blastocysts elongated). Conclusions Hand-made cloning is an efficient alternative for cloning in cattle. PMID:18205035

Black holes are almost optimal quantum cloners

NASA Astrophysics Data System (ADS)

Adami, Christoph; Ver Steeg, Greg

2015-06-01

If black holes were able to clone quantum states, a number of paradoxes in black hole physics would disappear. However, the linearity of quantum mechanics forbids exact cloning of quantum states. Here we show that black holes indeed clone incoming quantum states with a fidelity that depends on the black hole’s absorption coefficient, without violating the no-cloning theorem because the clones are only approximate. Perfectly reflecting black holes are optimal universal ‘quantum cloning machines’ and operate on the principle of stimulated emission, exactly as their quantum optical counterparts. In the limit of perfect absorption, the fidelity of clones is only equal to what can be obtained via quantum state estimation methods. But for any absorption probability less than one, the cloning fidelity is nearly optimal as long as ω /T≥slant 10, a common parameter for modest-sized black holes.

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

PubMed

Codner, Gemma F; Lindner, Loic; Caulder, Adam; Wattenhofer-Donzé, Marie; Radage, Adam; Mertz, Annelyse; Eisenmann, Benjamin; Mianné, Joffrey; Evans, Edward P; Beechey, Colin V; Fray, Martin D; Birling, Marie-Christine; Hérault, Yann; Pavlovic, Guillaume; Teboul, Lydia

2016-08-05

Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).

Rooting of conifer propagules

Treesearch

R.L. Mott

1977-01-01

An outline of the general problems involved with the propagation of elite conifer clones by rooted cuttings is drawn from published reports. New approaches for resolving these problems can come from studies of clone production through tissue culture methods. Probable extension of tissue culture techniques will permit the establishment of clones from adult, proven trees...

Cloning and study of the pectate lyase gene of Erwinia carotovora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.

1986-04-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector lambda 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representativemore » gene libraries on phage vectors from no less than 1 ..mu..g of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, lambda 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it.« less

Rapid, quantitative determination of bacteria in water. [adenosine triphosphate

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Picciolo, G. L.; Thomas, R. R.; Jeffers, E. L.; Deming, J. W. (Inventor)

1978-01-01

A bioluminescent assay for ATP in water borne bacteria is made by adding nitric acid to a water sample with concentrated bacteria to rupture the bacterial cells. The sample is diluted with sterile, deionized water, then mixed with a luciferase-luciferin mixture and the resulting light output of the bioluminescent reaction is measured and correlated with bacteria present. A standard and a blank also are presented so that the light output can be correlated to bacteria in the sample and system noise can be substracted from the readings. A chemiluminescent assay for iron porphyrins in water borne bacteria is made by adding luminol reagent to a water sample with concentrated bacteria and measuring the resulting light output of the chemiluminescent reaction.

Simplified methods for the construction of RNA and DNA virus infectious clones.

PubMed

Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

2015-01-01

Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

[Investigation of bacterial diversity in the biological desulfurization reactor for treating high salinity wastewater by the 16S rDNA cloning method].

PubMed

Liu, Wei-Guo; Liang, Cun-Zhen; Yang, Jin-Sheng; Wang, Gui-Ping; Liu, Miao-Miao

2013-02-01

The bacterial diversity in the biological desulfurization reactor operated continuously for 1 year was studied by the 16S rDNA cloning and sequencing method. Forty clones were randomly selected and their partial 16S rDNA genes (ca. 1,400 bp) were sequenced and blasted. The results indicated that there were dominant bacterias in the biological desulfurization reactor, where 33 clones belonged to 3 different published phyla, while 1 clone belonged to unknown phylum. The dominant bacterial community in the system was Proteobacteria, which accounted for 85.3%. The bacterial community succession was as follows: the gamma-Proteobacteria(55.9%), beta-Proteobacteria(17.6%), Actinobacteridae (8.8%), delta-Proteobacteria (5.9%) , alpha-Proteobacteria(5.9%), and Sphingobacteria (2.9%). Halothiobacillus sp. ST15 and Thiobacillus sp. UAM-I were the major desulfurization strains.

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

PubMed

Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

2015-11-01

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLC.

PubMed

Gaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M; Schnabel, Philipp A; Vollmer, Ekkehard; Reck, Martin; Goldmann, Torsten

2014-09-17

Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. EGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p=0.001). Overall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p=0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_165.

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

PubMed

Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

2012-12-01

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

Overview of post Cohen-Boyer methods for single segment cloning and for multisegment DNA assembly

PubMed Central

Sands, Bryan; Brent, Roger

2016-01-01

In 1973, Cohen and coworkers published a foundational paper describing the cloning of DNA fragments into plasmid vectors. In it, they used DNA segments made by digestion with restriction enzymes and joined these in vitro with DNA ligase. These methods established working recombinant DNA technology and enabled the immediate start of the biotechnology industry. Since then, “classical” recombinant DNA technology using restriction enzymes and DNA ligase has matured. At the same time, researchers have developed numerous ways to generate large, complex, multisegment DNA constructions that offer advantages over classical techniques. Here, we provide an overview of “post-Cohen-Boyer” techniques used for cloning single segments into vectors (T/A, Topo cloning, Gateway and Recombineering) and for multisegment DNA assembly (Biobricks, Golden Gate, Gibson, Yeast homologous recombination in vivo, and Ligase Cycling Reaction). We compare and contrast these methods and also discuss issues that researchers should consider before choosing a particular multisegment DNA assembly method. PMID:27152131

Methods of rapid, early selection of poplar clones for maximum yield potential: a manual of procedures.

Treesearch

USDA FS

1982-01-01

Instructions, illustrated with examples and experimental results, are given for the controlled-environment propagation and selection of poplar clones. Greenhouse and growth-room culture of poplar stock plants and scions are described, and statistical techniques for discriminating among clones on the basis of growth variables are emphasized.

Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis M-135

NASA Astrophysics Data System (ADS)

Yoshikazu, Kawata; Shin-Ichi, Yano; Hiroyuki, Kojima

1998-03-01

An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends with Taq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase gene crt B from Spirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

Human cloning: category, dignity, and the role of bioethics.

PubMed

Shuster, Evelyne

2003-10-01

Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

High-dimensional quantum cloning and applications to quantum hacking

PubMed Central

Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W.; Karimi, Ebrahim

2017-01-01

Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography. PMID:28168219

High-dimensional quantum cloning and applications to quantum hacking.

PubMed

Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W; Karimi, Ebrahim

2017-02-01

Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

PubMed

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

PubMed Central

Jacobus, Ana Paula; Gross, Jeferson

2015-01-01

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories. PMID:25774528

Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

PubMed

Manzano, Mark; Padmanabhan, Radhakrishnan

2014-01-01

The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

Tissue-Culture Method of Cloning Rubber Plants

NASA Technical Reports Server (NTRS)

Ball, E. A.

1983-01-01

Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

Human Cloning: Let's Discuss It.

ERIC Educational Resources Information Center

Taras, Loretta; Stavroulakis, Anthea M.; Ortiz, Mary T.

1999-01-01

Describes experiences with holding discussions on cloning at a variety of levels in undergraduate biology courses. Discusses teaching methods used and student reactions to the discussions. Contains 12 references. (WRM)

Reproductive ability of a cloned male detector dog and behavioral traits of its offspring

PubMed Central

Lee, Ji Hyun; Kim, Geon A; Kim, Rak Seung; Lee, Jong Su; Oh, Hyun Ju; Kim, Min Jung; Hong, Do Kyo

2016-01-01

In 2007, seven detector dogs were produced by somatic cell nuclear transfer using one nuclear donor dog, then trained and certified as excellent detector dogs, similar to their donor. In 2011, we crossed a cloned male and normal female by natural breeding and produced ten offspring. In this study, we investigated the puppies' temperaments, which we later compared with those of the cloned parent male. The results show that the cloned male had normal reproductive abilities and produced healthy offspring. All puppies completed narcotic detector dog training with a success rate for selection of 60%. Although the litter of cloned males was small in this study, a cloned male dog bred by natural mating produced puppies that later successfully completed the training course for drug detection. In conclusion, cloning an elite dog with superior genetic factors and breeding of the cloned dog was found to be a useful method to efficiently procure detector dogs. PMID:26435541

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reproductive ability of a cloned male detector dog and behavioral traits of its offspring.

PubMed

Lee, Ji Hyun; Kim, Geon A; Kim, Rak Seung; Lee, Jong Su; Oh, Hyun Ju; Kim, Min Jung; Hong, Do Kyo; Lee, Byeong Chun

2016-09-30

In 2007, seven detector dogs were produced by somatic cell nuclear transfer using one nuclear donor dog, then trained and certified as excellent detector dogs, similar to their donor. In 2011, we crossed a cloned male and normal female by natural breeding and produced ten offspring. In this study, we investigated the puppies' temperaments, which we later compared with those of the cloned parent male. The results show that the cloned male had normal reproductive abilities and produced healthy offspring. All puppies completed narcotic detector dog training with a success rate for selection of 60%. Although the litter of cloned males was small in this study, a cloned male dog bred by natural mating produced puppies that later successfully completed the training course for drug detection. In conclusion, cloning an elite dog with superior genetic factors and breeding of the cloned dog was found to be a useful method to efficiently procure detector dogs.

Cloning cattle: the methods in the madness.

PubMed

Oback, Björn; Wells, David N

2007-01-01

Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.

Systematic cloning of an ORFeome using the Gateway system.

PubMed

Matsuyama, Akihisa; Yoshida, Minoru

2009-01-01

With the completion of the genome projects, there are increasing demands on the experimental systems that enable to exploit the entire set of protein-coding open reading frames (ORFs), viz. ORFeome, en masse. Systematic proteomic studies based on cloned ORFeomes are called "reverse proteomics," and have been launched in many organisms in recent years. Cloning of an ORFeome is such an attractive way for comprehensive understanding of biological phenomena, but is a challenging and daunting task. However, recent advances in techniques for DNA cloning using site-specific recombination and for high-throughput experimental techniques have made it feasible to clone an ORFeome with the minimum of exertion. The Gateway system is one of such the approaches, employing the recombination reaction of the bacteriophage lambda. Combining traditional DNA manipulation methods with modern technique of the recombination-based cloning system, it is possible to clone an ORFeome of an organism on an individual level.

Construction of an infectious genomic clone of porcine parvovirus: effect of the 5'-end on DNA replication.

PubMed

Casal, J I; Diaz-Aroca, E; Ranz, A I; Manclus, J J

1990-08-01

The linear single-stranded DNA genome of the porcine parvovirus, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pUC18 using a simple and reliable method. These clones were stable during propagation in Escherichia coli JM109. The recombinant clones of porcine parvovirus were infectious when transfected into monolayers of swine testes cells as identified by the development of cytopathic effect, indirect immunofluorescence with specific antiserum, and hemagglutination assays. DNA isolated from progeny virus arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. The presence of the turn of the 5'-end loop seems to be necessary to get stable infectious clones.

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

PubMed

Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

2002-12-01

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

PubMed

Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

2002-04-01

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

NASA Technical Reports Server (NTRS)

Ponnaiya, B.; Limoli, C. L.; Corcoran, J.; Kaplan, M. I.; Hartmann, A.; Morgan, W. F.

1998-01-01

PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

VizieR Online Data Catalog: NIBLES. I. The Nancay HI survey (van Driel+, 2016)

NASA Astrophysics Data System (ADS)

van Driel, M.; Butcher, Z.; Schneider, S.; Lehnert, M.; Minchin, R.; Blyth, S.-L.; Chemin, L.; Hallet, N.; Joseph, T.; Kotze, P.; Kraan-Korteweg, R. C.; Olofsson, H.; Ramatsoku, M.

2016-11-01

HI 21cm line spectra of the 1870 clearly or marginally detected SDSS sources obtained for NIBLES at the Nancay Radio Telescope. Please note that these include the six detections with velocities below the 900 km/s lower limit for the NIBLES statistical sample, which are listed in Table A.5 (NIBLES sources 0347, 1572, 1734, 1897, 2259, and 2326). See Sect. 3 of the paper for further details on data acquisition and reduction. Data have been smoothed in velocity to 18 km/s resolution (see exact number in the spectrum headers). A fitted polynomial baseline was substracted from the observed spectra. Velocities (first column) are heliocentric in the optical convention in units of km/s and flux densities (second column) are in Janskys. (4 data files).

High throughput generation and characterization of replication-competent clade C transmitter-founder simian human immunodeficiency viruses

PubMed Central

Dutta, Debashis; Johnson, Samuel; Dalal, Alisha; Deymier, Martin J.; Hunter, Eric

2018-01-01

Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies. PMID:29758076

Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

PubMed Central

Gay, Glen; Wagner, Drew T.; Keatinge-Clay, Adrian T.; Gay, Darren C.

2014-01-01

The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast. PMID:25304917

An Investigative Graduate Laboratory Course for Teaching Modern DNA Techniques

ERIC Educational Resources Information Center

de Lencastre, Alexandre; Torello, A. Thomas; Keller, Lani C.

2017-01-01

This graduate-level DNA methods laboratory course is designed to model a discovery-based research project and engages students in both traditional DNA analysis methods and modern recombinant DNA cloning techniques. In the first part of the course, students clone the "Drosophila" ortholog of a human disease gene of their choosing using…

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

PubMed

Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

2017-04-01

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Treatment of donor cell/embryo with different approaches to improve development after nuclear transfer.

PubMed

Mizutani, Eiji; Wakayama, Sayaka; Wakayama, Teruhiko

2015-01-01

The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. Amore » minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.« less

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).

PubMed

Hoskins, Roger A; Stapleton, Mark; George, Reed A; Yu, Charles; Wan, Kenneth H; Carlson, Joseph W; Celniker, Susan E

2005-12-02

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT-PCR approaches.

Factors influencing the commercialisation of cloning in the pork industry.

PubMed

Pratt, S L; Sherrer, E S; Reeves, D E; Stice, S L

2006-01-01

Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.

Paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti-thymocyte globulin plus cyclosporine

PubMed Central

Scheinberg, Phillip; Marte, Michael; Nunez, Olga; Young, Neal S.

2010-01-01

Background Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40–50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia. Design and Methods We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a horse anti-thymocyte globulin plus cyclosporine regimen from 2000 to 2008. Results The presence of a glycosylphosphatidylinositol-anchor protein-deficient clone was detected in 83 (40%) patients pre-treatment, and the median clone size was 9.7% (interquartile range 3.5–29). In patients without a detectable clone pre-treatment, the appearance of a clone after immunosuppressive therapy was infrequent, and in most with a clone pre-treatment, clone size often decreased after immunosuppressive therapy. However, in 30 patients, an increase in clone size was observed after immunosuppressive therapy. The majority of patients with a paroxysmal nocturnal hemoglobinuria clone detected after immunosuppressive therapy did not have an elevated lactate dehydrogenase, nor did they experience hemolysis or thrombosis, and they did not require specific interventions with anticoagulation and/or eculizumab. Of the 7 patients who did require therapy for clinical paroxysmal nocturnal hemoglobinuria symptoms and signs, all had an elevated lactate dehydrogenase and a clone size greater than 50%. In all, 18 (8.6%) patients had a clone greater than 50% at any given time of sampling. Conclusions The presence of a paroxysmal nocturnal hemoglobinuria clone in severe aplastic anemia is associated with low morbidity and mortality, and specific measures to address clinical paroxysmal nocturnal hemoglobinuria are seldom required. PMID:20595102

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

HIGH-THROUGHPUT IDENTIFICATION OF THE PREDOMINANT MALARIA PARASITE CLONE IN COMPLEX BLOOD STAGE INFECTIONS USING A MULTI-SNP MOLECULAR HAPLOTYPING ASSAY

PubMed Central

COLE-TOBIAN, JENNIFER L.; ZIMMERMAN, PETER A.; KING, CHRISTOPHER L.

2013-01-01

Individuals living in malaria endemic areas are often infected with multiple parasite clones. Currently used single nucleotide polymorphism (SNP) genotyping methods for malaria parasites are cumbersome; furthermore, few methods currently exist that can rapidly determine the most abundant clone in these complex infections. Here we describe an oligonucleotide ligation assay (OLA) to distinguish SNPs in the Plasmodium vivax Duffy binding protein gene (Pvdbp) at 14 polymorphic residues simultaneously. Allele abundance is determined by the highest mean fluorescent intensity of each allele. Using mixtures of plasmids encoding known haplotypes of the Pvdbp, single clones of P. vivax parasites from infected Aotus monkeys, and well-defined mixed infections from field samples, we were able to identify the predominant Pvdbp genotype with > 93% accuracy when the dominant clone is twice as abundant as a lesser genotype and > 97% of the time if the ratio was 5:1 or greater. Thus, the OLA can accurately, reproducibly, and rapidly determine the predominant parasite haplotype in complex blood stage infections. PMID:17255222

Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.

PubMed

Kuo, Jonathan; Inman, Jason; Brownstein, Michael; Usdin, Ted B

2004-12-15

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Orpheus recombination : a comprehensive bacteriophage system for murine targeting vector construction by transplacement.

PubMed

Woltjen, Knut; Ito, Kenichi; Tsuzuki, Teruhisa; Rancourt, Derrick E

2008-01-01

In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous "probe" region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.

ddClone: joint statistical inference of clonal populations from single cell and bulk tumour sequencing data.

PubMed

Salehi, Sohrab; Steif, Adi; Roth, Andrew; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

2017-03-01

Next-generation sequencing (NGS) of bulk tumour tissue can identify constituent cell populations in cancers and measure their abundance. This requires computational deconvolution of allelic counts from somatic mutations, which may be incapable of fully resolving the underlying population structure. Single cell sequencing (SCS) is a more direct method, although its replacement of NGS is impeded by technical noise and sampling limitations. We propose ddClone, which analytically integrates NGS and SCS data, leveraging their complementary attributes through joint statistical inference. We show on real and simulated datasets that ddClone produces more accurate results than can be achieved by either method alone.

[Scientific ethics of human cloning].

PubMed

Valenzuela, Carlos Y

2005-01-01

True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

Positional cloning in maize (Zea mays subsp. mays, Poaceae)1

PubMed Central

Gallavotti, Andrea; Whipple, Clinton J.

2015-01-01

• Premise of the study: Positional (or map-based) cloning is a common approach to identify the molecular lesions causing mutant phenotypes. Despite its large and complex genome, positional cloning has been recently shown to be feasible in maize, opening up a diverse collection of mutants to molecular characterization. • Methods and Results: Here we outline a general protocol for positional cloning in maize. While the general strategy is similar to that used in other plant species, we focus on the unique resources and approaches that should be considered when applied to maize mutants. • Conclusions: Positional cloning approaches are appropriate for maize mutants and quantitative traits, opening up to molecular characterization the large array of genetic diversity in this agronomically important species. The cloning approach described should be broadly applicable to other species as more plant genomes become available. PMID:25606355

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

PubMed Central

Oh, Man Hwan; Lee, Je Chul; Kim, Jungmin

2015-01-01

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii. PMID:25746991

Molecular analysis of microflora associated with dentoalveolar abscesses.

PubMed Central

Dymock, D; Weightman, A J; Scully, C; Wade, W G

1996-01-01

The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned. Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria. Clones representative of five predominant groups of uncultured organisms were sequenced. Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros. The remaining two clones did not correspond to known, previously sequenced organisms. One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella. This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture. PMID:8904410

DNA cloning: A personal view after 40 years

PubMed Central

Cohen, Stanley N.

2013-01-01

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer’s laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning. PMID:24043817

A novel cloning template designing method by using an artificial bee colony algorithm for edge detection of CNN based imaging sensors.

PubMed

Parmaksızoğlu, Selami; Alçı, Mustafa

2011-01-01

Cellular Neural Networks (CNNs) have been widely used recently in applications such as edge detection, noise reduction and object detection, which are among the main computer imaging processes. They can also be realized as hardware based imaging sensors. The fact that hardware CNN models produce robust and effective results has attracted the attention of researchers using these structures within image sensors. Realization of desired CNN behavior such as edge detection can be achieved by correctly setting a cloning template without changing the structure of the CNN. To achieve different behaviors effectively, designing a cloning template is one of the most important research topics in this field. In this study, the edge detecting process that is used as a preliminary process for segmentation, identification and coding applications is conducted by using CNN structures. In order to design the cloning template of goal-oriented CNN architecture, an Artificial Bee Colony (ABC) algorithm which is inspired from the foraging behavior of honeybees is used and the performance analysis of ABC for this application is examined with multiple runs. The CNN template generated by the ABC algorithm is tested by using artificial and real test images. The results are subjectively and quantitatively compared with well-known classical edge detection methods, and other CNN based edge detector cloning templates available in the imaging literature. The results show that the proposed method is more successful than other methods.

A Novel Cloning Template Designing Method by Using an Artificial Bee Colony Algorithm for Edge Detection of CNN Based Imaging Sensors

PubMed Central

Parmaksızoğlu, Selami; Alçı, Mustafa

2011-01-01

Cellular Neural Networks (CNNs) have been widely used recently in applications such as edge detection, noise reduction and object detection, which are among the main computer imaging processes. They can also be realized as hardware based imaging sensors. The fact that hardware CNN models produce robust and effective results has attracted the attention of researchers using these structures within image sensors. Realization of desired CNN behavior such as edge detection can be achieved by correctly setting a cloning template without changing the structure of the CNN. To achieve different behaviors effectively, designing a cloning template is one of the most important research topics in this field. In this study, the edge detecting process that is used as a preliminary process for segmentation, identification and coding applications is conducted by using CNN structures. In order to design the cloning template of goal-oriented CNN architecture, an Artificial Bee Colony (ABC) algorithm which is inspired from the foraging behavior of honeybees is used and the performance analysis of ABC for this application is examined with multiple runs. The CNN template generated by the ABC algorithm is tested by using artificial and real test images. The results are subjectively and quantitatively compared with well-known classical edge detection methods, and other CNN based edge detector cloning templates available in the imaging literature. The results show that the proposed method is more successful than other methods. PMID:22163903

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

PubMed

Shu, Changlong; Zhang, Jingtao; Chen, Guihua; Liang, Gemei; He, Kanglai; Crickmore, Neil; Huang, Dafang; Zhang, Jie; Song, Fuping

2013-09-01

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

PubMed

Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

2017-11-14

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

[Archaeal community structure and diversity in Urumqi No. 10 cold sulfur spring analyzed by culture-independent approach].

PubMed

Li, Ping; Zeng, Jun; Zulipiya, Yunus; Gao, Xiaoqi; Dong, Xiuhuang; Xue, Juan; Lou, Kai

2013-03-04

We explored the composition and diversity of archaea in a cold sulfur spring water in Xinjiang earthquake fault zone. Environmental total DNA was extracted directly with enzymatic lysis method from a cold sulfur spring water. We constructed clone library of 16S rRNA gene amplified with archaeal-specific primers. A total of 115 positive clones were selected randomly from the library and identified by restriction length polymorphism (RFLP) with enzyme Alu I and Afa I. The unique RFLP patterns corresponded clones were selected for sequencing, BLAS alignment and constructing 16S rRNA gene phylogenetic tree. In total, 44 operational taxonomic units (OTUs) were determined from the library. BLAST and phylogenetic analysis indicated that these OTUs were affiliated with Euryarchaeota (94.78%) and Thaumarchaeota (4.35%). Only one Thaumarchaeotal clone was detected and most related to the genus Nitrosopumilus with 93% similarity. Euryarchaeotal clones were abundant and diverse. Of them, 42.61% of clones belonged to RC-V cluster; 13.91% of clones, 20.87% of clones were classified into LDS cluster and Methanomicrobiales respectively; 4.35% of clones had high similarity with ANME-1a-FW, which were involved in Anaerobic oxidation of methane (AOM). In addition, we also detected some (13.05%) unknown Euryarchaotal clones. Euryarchaeota in the environment were diverse, and possibly with a large fraction of potential novel species.

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

Rise and fall of outbreak-specific clone inside endemic pulsotype of Salmonella 4,[5],12:i:-; insights from high-resolution molecular surveillance in Emilia-Romagna, Italy, 2012 to 2015.

PubMed

Morganti, Marina; Bolzoni, Luca; Scaltriti, Erika; Casadei, Gabriele; Carra, Elena; Rossi, Laura; Gherardi, Paola; Faccini, Fabio; Arrigoni, Norma; Sacchi, Anna Rita; Delledonne, Marco; Pongolini, Stefano

2018-03-01

Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions : The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

PubMed

Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

2016-12-01

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

PubMed

Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

2009-09-01

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

A strategy for rapid production and screening of yeast artificial chromosome libraries.

PubMed

Strauss, W M; Jaenisch, E; Jaenisch, R

1992-01-01

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

Wavelength interrogation of fiber Bragg grating sensors based on crossed optical Gaussian filters.

PubMed

Cheng, Rui; Xia, Li; Zhou, Jiaao; Liu, Deming

2015-04-15

Conventional intensity-modulated measurements require to be operated in linear range of filter or interferometric response to ensure a linear detection. Here, we present a wavelength interrogation system for fiber Bragg grating sensors where the linear transition is achieved with crossed Gaussian transmissions. This unique filtering characteristic makes the responses of the two branch detections follow Gaussian functions with the same parameters except for a delay. The substraction of these two delayed Gaussian responses (in dB) ultimately leads to a linear behavior, which is exploited for the sensor wavelength determination. Beside its flexibility and inherently power insensitivity, the proposal also shows a potential of a much wider operational range. Interrogation of a strain-tuned grating was accomplished, with a wide sensitivity tuning range from 2.56 to 8.7 dB/nm achieved.

Self-calibrating threshold detector

NASA Technical Reports Server (NTRS)

Barnes, J. R.; Huang, M. Y. (Inventor)

1980-01-01

A self calibrating threshold detector comprises a single demodulating channel which includes a mixer having one input receiving the incoming signal and another input receiving a local replica code. During a short time interval, an incorrect local code is applied to the mixer to incorrectly demodulate the incoming signal and to provide a reference level that calibrates the noise propagating through the channel. A sample and hold circuit is coupled to the channel for storing a sample of the reference level. During a relatively long time interval, the correct replica code provides an output level which ranges between the reference level and a maximum level that represents incoming signal presence and synchronism with the replica code. A summer substracts the stored sample reference from the output level to provide a resultant difference signal indicative of the acquisition of the expected signal.

Cloning in America: constitutional rights and limits.

PubMed

Erwin, C

2000-01-01

As readers of science fiction are well aware, the term "clone" refers to asexually produced offspring, that is, produced by a process of cell-division which does not begin with the union of two sex cells. A clone would be the genetic twin of the cell donor. Propagation of plants by this method is, of course, commonplace, but mammalian reproduction in this fashion would be indeed a revolutionary accomplishment, with profound and disturbing implications.

[Human cloning or cannibalism].

PubMed

Sokolowski, L M

2001-01-01

In this article I develop the idea presented in my previous work that human cloning would be of little practical use since almost any aim that one would like to attain by multiple cloning of a concrete man or a group of people, are unattainable or it might be achieved by easier, cheaper and more efficient traditional methods. For this reason cloning of a man is unlikely to occur on a larger scale and only few people will decide to clone themselves. In this sense no social effects of human cloning will be disastrous for the human population. Yet investigations in human genetics are very important since they may provide medical applications far more important than human cloning. It is argued that the main trend of modern medicine: organ transplantation from an alien donor, will become socially dangerous in near future since the number of donors will be drastically smaller than the number of potential patients waiting for transplantations. This in turn may cause social conflicts and a form of medical cannibalism may arise. These problems and conflicts will be avoided if organ transplantation from an alien donor is replaced by organ cloning, i.e. by transplanting an organ developed from the patient.

pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students

PubMed Central

Eckdahl, Todd; Cronk, Brian; Andresen, Corinne; Frederick, Paul; Huckuntod, Samantha; Shinneman, Claire; Wacker, Annie; Yuan, Jason

2014-01-01

The Vision and Change report recommended genuine research experiences for undergraduate biology students. Authentic research improves science education, increases the number of scientifically literate citizens, and encourages students to pursue research. Synthetic biology is well suited for undergraduate research and is a growing area of science. We developed a laboratory module called pClone that empowers students to use advances in molecular cloning methods to discover new promoters for use by synthetic biologists. Our educational goals are consistent with Vision and Change and emphasize core concepts and competencies. pClone is a family of three plasmids that students use to clone a new transcriptional promoter or mutate a canonical promoter and measure promoter activity in Escherichia coli. We also developed the Registry of Functional Promoters, an open-access database of student promoter research results. Using pre- and posttests, we measured significant learning gains among students using pClone in introductory biology and genetics classes. Student posttest scores were significantly better than scores of students who did not use pClone. pClone is an easy and affordable mechanism for large-enrollment labs to meet the high standards of Vision and Change. PMID:26086659

Endovascular treatment of ruptured true posterior communicating artery aneurysms.

PubMed

Yang, Yonglin; Su, Wandong; Meng, Qinghai

2015-01-01

Although true posterior communicating artery (PCoA) aneurysms are rare, they are of vital importance. We reviewed 9 patients with this fatal disease, who were treated with endovascular embolization, and discussed the meaning of endovascular embolization for the treatment of true PCoA aneurysms. From September 2006 to May 2012, 9 patients with digital substraction angiography (DSA) confirmed true PCoA aneurysms were treated with endovascular embolization. Patients were followed-up with a minimal duration of 17 months and assessed by Glasgow Outcome Scale (GOS) score. All the patients presented with spontaneous subarachnoid hemorrhage from the ruptured aneurysms. The ratio of males to females was 1:2, and the average age of onset was 59.9 (ranging from 52 to 72) years. The preoperative Hunt-Hess grade scores were I to III. All patients had recovered satisfactorily. No permanent neurological deficits were left. Currently, endovascular embolization can be recommended as the top choice for the treatment of most true PCoA aneurysms, due to its advanced technique, especially the application of the stent-assisted coiling technique, combined with its advantage of mininal invasiveness and quick recovery. However, the choice of treatment methods should be based on the clinical and anatomical characteristics of the aneurysm and the skillfulness of the surgeon.

The high diversity of MRSA clones detected in a university hospital in istanbul.

PubMed

Oksuz, Lutfiye; Dupieux, Celine; Tristan, Anne; Bes, Michele; Etienne, Jerome; Gurler, Nezahat

2013-01-01

To characterize the methicillin-resistant Staphylococcus aureus (MRSA) clones present in Istanbul, 102 MRSA isolates collected during a 5-year period at the Istanbul Medical Faculty Hospital were characterized using microarray analysis and phenotypic resistance profiles. Resistance to methicillin was detected with a cefoxitin disk diffusion assay and confirmed with a MRSA-agar and MRSA detection kit. Antimicrobial susceptibility testing was performed by a disk diffusion assay and interpreted according to the 2012 guidelines of the Antibiogram Committee of the French Society for Microbiology. Decreased susceptibility to glycopeptides was confirmed using the population analysis profile-area under the curve (PAP-AUC) method. The presence of the mecA gene was detected by polymerase chain reaction. Bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits. Strains were extensively characterized using the DNA microarray. Isolates were grouped into six clonal complexes. The most frequently detected clone was the Vienna/Hungarian/Brazilian clone (ST239-MRSA-III), which accounted for 53.9% of the isolates. These isolates were resistant to multiple antibiotics, particularly penicillin, tetracycline, rifampicin, kanamycin, tobramycin, gentamicin, levofloxacin, erythromycin, lincomycin and fosfomycin. Furthermore, three isolates were detected by population analysis profile as heterogeneous vancomycin-intermediate S. aureus (hVISA). The UK-EMRSA-15 clone (ST22-MRSA-IV PVL negative) was detected in 9.8% of the isolates and was mainly susceptible to all anti-staphylococcal antibiotics. Seven isolates (6.9%) were positive for PVL genes and were assigned to the CC80-MRSA-IV clone (European CA-MRSA clone, three isolates), ST8-MRSA-IV clone (USA300 clone, two isolates, one ACME-positive) or ST22-MRSA-IV clone ("Regensburg EMRSA" clone, two isolates). All other clones were detected in one to six isolates and corresponded to well-known clones (e.g., Pediatric clone, Dublin EMRSA clone, WA MRSA-54/63, WA MRSA-1/57). This work highlighted both the high prevalence of ST239-MRSA-III clone and the large diversity of the other MRSA clones detected in a university hospital in Istanbul.

The potential for modification in cloning and vitrification technology to enhance genetic progress in beef cattle in Northern Australia.

PubMed

Taylor-Robinson, Andrew W; Walton, Simon; Swain, David L; Walsh, Kerry B; Vajta, Gábor

2014-08-01

Recent advances in embryology and related research offer considerable possibilities to accelerate genetic improvement in cattle breeding. Such progress includes optimization and standardization of laboratory embryo production (in vitro fertilization - IVF), introduction of a highly efficient method for cryopreservation (vitrification), and dramatic improvement in the efficiency of somatic cell nuclear transfer (cloning) in terms of required effort, cost, and overall outcome. Handmade cloning (HMC), a simplified version of somatic cell nuclear transfer, offers the potential for relatively easy and low-cost production of clones. A potentially modified method of vitrification used at a centrally located laboratory facility could result in cloned offspring that are economically competitive with elite animals produced by more traditional means. Apart from routine legal and intellectual property issues, the main obstacle that hampers rapid uptake of these technologies by the beef cattle industry is a lack of confidence from scientific and commercial sources. Once stakeholder support is increased, the combined application of these methods makes a rapid advance toward desirable traits (rapid growth, high-quality beef, optimized reproductive performance) a realistic goal. The potential impact of these technologies on genetic advancement in beef cattle herds in which improvement of stock is sought, such as in northern Australia, is hard to overestimate. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

DNA probe for lactobacillus delbrueckii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delley, M.; Mollet, B.; Hottinger, H.

1990-06-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

DNA Probe for Lactobacillus delbrueckii

PubMed Central

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

Monitoring of microbial communities in anaerobic digestion sludge for biogas optimisation.

PubMed

Lim, Jun Wei; Ge, Tianshu; Tong, Yen Wah

2018-01-01

This study characterised and compared the microbial communities of anaerobic digestion (AD) sludge using three different methods - (1) Clone library; (2) Pyrosequencing; and (3) Terminal restriction fragment length polymorphism (T-RFLP). Although high-throughput sequencing techniques are becoming increasingly popular and affordable, the reliance of such techniques for frequent monitoring of microbial communities may be a financial burden for some. Furthermore, the depth of microbial analysis revealed by high-throughput sequencing may not be required for monitoring purposes. This study aims to develop a rapid, reliable and economical approach for the monitoring of microbial communities in AD sludge. A combined approach where genetic information of sequences from clone library was used to assign phylogeny to T-RFs determined experimentally was developed in this study. In order to assess the effectiveness of the combined approach, microbial communities determined by the combined approach was compared to that characterised by pyrosequencing. Results showed that both pyrosequencing and clone library methods determined the dominant bacteria phyla to be Proteobacteria, Firmicutes, Bacteroidetes, and Thermotogae. Both methods also found that sludge A and B were predominantly dominated by acetogenic methanogens followed by hydrogenotrophic methanogens. The number of OTUs detected by T-RFLP was significantly lesser than that detected by the clone library. In this study, T-RFLP analysis identified majority of the dominant species of the archaeal consortia. However, many of the more highly diverse bacteria consortia were missed. Nevertheless, the combined approach developed in this study where clone sequences from the clone library were used to assign phylogeny to T-RFs determined experimentally managed to accurately predict the same dominant microbial groups for both sludge A and sludge B, as compared to the pyrosequencing results. Results showed that the combined approach of clone library and T-RFLP accurately predicted the dominant microbial groups and thus is a reliable and more economical way to monitor the evolution of microbial systems in AD sludge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of positive control materials for DNA-based detection of cystic fibrosis: Cloning and sequencing of 31 mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iovannisci, D.; Brown, C.; Winn-Deen, E.

1994-09-01

The cloning and sequencing of the gene associated with cystic fibrosis (CF) now provides the opportunity for earlier detection and carrier screening through DNA-based detection schemes. To date, over 300 mutations have been reported to the CF Consortium; however, only 30 mutations have been observed frequently enough world-wide to warrant routine screening. Many of these mutations are not available as cloned material or as established tissue culture cell lines to aid in the development of DNA-based detection assays. We have therefore cloned the 30 most frequently reported mutations, plus the mutation R347H due to its association with male infertility (31more » mutations, total). Two approaches were employed: direct PCR amplification, where mutations were available from patient sources, and site-directed PCR mutagenesis of normal genomic DNA to generate the remaining mutations. After amplification, products were cloned into a sequencing vector, bacterial transformants were screened by a novel method (PCR/oligonucleotide litigation assay/sequence-coded separation), and plamid DNA sequences determined by automated fluorescent methods on the Applied Biosystems 373A. Mixing of the clones allows the construction of artificial genotypes useful as positive control material for assay validation. A second round of mutagenesis, resulting in the construction of plasmids bearing multiple mutations, will be evaluated for their utility as reagent control materials in kit development.« less

A BAC clone fingerprinting approach to the detection of human genome rearrangements

PubMed Central

Krzywinski, Martin; Bosdet, Ian; Mathewson, Carrie; Wye, Natasja; Brebner, Jay; Chiu, Readman; Corbett, Richard; Field, Matthew; Lee, Darlene; Pugh, Trevor; Volik, Stas; Siddiqui, Asim; Jones, Steven; Schein, Jacquie; Collins, Collin; Marra, Marco

2007-01-01

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements. PMID:17953769

Cloning the Gravity and Shear Stress Related Genes from MG-63 Cells by Subtracting Hybridization

NASA Astrophysics Data System (ADS)

Zhang, Shu; Dai, Zhong-quan; Wang, Bing; Cao, Xin-sheng; Li, Ying-hui; Sun, Xi-qing

2008-06-01

Background The purpose of the present study was to clone the gravity and shear stress related genes from osteoblast-like human osteosarcoma MG-63 cells by subtractive hybridization. Method MG-63 cells were divided into two groups (1G group and simulated microgravity group). After cultured for 60 h in two different gravitational environments, two groups of MG-63 cells were treated with 1.5Pa fluid shear stress (FSS) for 60 min, respectively. The total RNA in cells was isolated. The gravity and shear stress related genes were cloned by subtractive hybridization. Result 200 clones were gained. 30 positive clones were selected using PCR method based on the primers of vector and sequenced. The obtained sequences were analyzed by blast. changes of 17 sequences were confirmed by RT-PCR and these genes are related to cell proliferation, cell differentiation, protein synthesis, signal transduction and apoptosis. 5 unknown genes related to gravity and shear stress were found. Conclusion In this part of our study, our result indicates that simulated microgravity may change the activities of MG-63 cells by inducing the functional alterations of specific genes.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

PubMed

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Comparative Analysis of Growth and Photosynthetic Characteristics of (Populus simonii × P. nigra) × (P. nigra × P. simonii) Hybrid Clones of Different Ploidides

PubMed Central

Bian, Xiuyan; Liu, Mengran; Sun, Yanshuang; Jiang, Jing; Wang, Fuwei; Li, Shuchun; Cui, Yonghong; Liu, Guifeng; Yang, Chuanping

2015-01-01

To evaluate differences among poplar clones of various ploidies, 12 hybrid poplar clones (P. simonii × P. nigra) × (P. nigra × P. simonii) with different ploidies were used to study phenotypic variation in growth traits and photosynthetic characteristics. Analysis of variance showed remarkable differences for each of the investigated traits among these clones (P < 0.01). Coefficients of phenotypic variation (PCV) ranged from 2.38% to 56.71%, and repeatability ranged from 0.656 to 0.987. The Pn (photosynthetic rate) photosynthetic photon flux density (PPFD) curves of the 12 clones were S-shaped, but the Pn-ambient CO2 (Ca) curves were shaped like an inverted “V”. The stomatal conductance (Gs)-PPFD and transpiration rate (Tr)-PPFD curves had an upward tendency; however, with increasing PFFD, the intercellular CO2 concentration (Ci)-PPFD curves had a downward tendency in all of the clones. The Pn-PPFD and Pn-Ca curves followed the pattern of a quadratic equation. The average light saturation point and light compensation point of the triploid clones were the highest and lowest, respectively, among the three types of clones. For Pn-Ca curves, diploid clones had a higher average CO2 saturation point and average CO2 compensation point compared with triploid and tetraploid clones. Correlation analyses indicated that all investigated traits were strongly correlated with each other. In future studies, molecular methods should be used to analyze poplar clones of different ploidies to improve our understanding of the growth and development mechanisms of polyploidy. PMID:25867100

Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

PubMed

Singh, K M; Tripathi, A K; Pandya, P R; Rank, D N; Kothari, R K; Joshi, C G

2011-09-01

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

SPE-HPTLC of procyanidins from the barks of different species and clones of Salix.

PubMed

Pobłocka-Olech, Loretta; Krauze-Baranowska, Mirosława

2008-11-04

A SPE-HPTLC method was developed for the qualitative and quantitative analysis of procyanidin B(1) in willow barks. The chromatography was performed on HPTLC silica gel layer with the mobile phase chloroform-ethanol-formic acid (50:40:6 v/v/v), in the Automatic Developing Chamber-ADC 2. The methanol extracts from willow barks were purified by SPE method on RP-18 silica gel columns with methanol-water (7:93 v/v) as the eluent. The presence of procyanidin B(1) was revealed in the majority of investigated willow barks. The content of procyanidin B(1) varied from 0.26 mg/g in the extract of Salix purpurea clone 1067-2.24 mg/g in the extract of Salix alba clone 1100. The method was validated for linearity, precision, LOD, LOQ and repeatability.

Quality Evalution of Potato Clones as Processed Material Cultivated in Lembang

NASA Astrophysics Data System (ADS)

Rahayu, S. T.; Handayani, T.; Levianny, P. S.

2017-03-01

Potatoes are widely grown in the temperate as well as tropical zones and are the fourth largest staple crop in the world after maize, wheat and rice. The study aimed to evaluate the quality of several potato clones as raw material on potato based products (chips and boiled). The study was conducted at Indonesian Vegetable Research Institute, Lembang about 1200 m asl height, in 2016. The design used was a randomized complete block design with three replications. The samples tested were 5 clones selection (clones number 1,2,3,4,10). In this study, variety Granola (Clone number 6) and Atlantic (Clone number 7) were used as a susceptible control, meanwhile the Katahdin (Clone number 8) and SP 951 (Clone number 9) were used as the resistant control. Chemical properties tested were starch, reduction sugar, water content, specific gravity, and Total Soluble Solute (TSS). The organoleptic assessment method used was hedonic test with scale of 1-5 (very like until very dislike) which had been done by 15 untrained panelists. Data was statisticaly analized by Duncan’s test (5%). Clone 1 and 2 were preferred by panelist as raw material for potato chips, which got score of ‘very like’ until ‘like’ for color, size, taste, and texture parameters. Although there was no significant difference on color and size parameters for all samples of that boiled potato there, however, clone no 8 can be considered as the most favourite based on taste and texture parameters.

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

PubMed

Kim, Eunhye; Zheng, Zhong; Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

2016-01-01

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimelman, Aya; Levy, Asaf; Sberro, Hila

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of smallmore » non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.« less

Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years.

PubMed

Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko

2008-11-11

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 degrees C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.

Production of healthy cloned mice from bodies frozen at −20°C for 16 years

PubMed Central

Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko

2008-01-01

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the “resurrection” of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at −20 °C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to “resurrect” animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation. PMID:18981419

Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

PubMed

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

2015-03-20

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

PubMed

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

PubMed Central

Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

2012-01-01

Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

PubMed

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Tanomand, Asghar; Akbari, Bahman

2016-11-01

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Cloning: Learning to Replay the Genetic Tape.

ERIC Educational Resources Information Center

Holden, David J.

1979-01-01

Describes how plants can be produced by cloning by using tissue culture methods to mass-produce rare native prairie plants and trying to transfer some of the genetic characteristics of native grasses into cultivated cereals. The experiment was conducted at South Dakota State University. (HM)

Large-Scale Concatenation cDNA Sequencing

PubMed Central

Yu, Wei; Andersson, Björn; Worley, Kim C.; Muzny, Donna M.; Ding, Yan; Liu, Wen; Ricafrente, Jennifer Y.; Wentland, Meredith A.; Lennon, Greg; Gibbs, Richard A.

1997-01-01

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching. [All 65 cDNA clone sequences described in this paper have been submitted to the GenBank data library under accession nos. U79240–U79304.] PMID:9110174

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

PubMed

Hara, H; Kaji, H

1987-02-01

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

Isolation and characterization of 21 novel expressed DNA sequences from the distal region of human chromosome 4p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Yoshikazu; Hadano, Shinji; Nagayama, Tomiko

1994-07-15

The authors have established an approach to the isolation of expressed DNA sequences from a defined region of the human chromosome. The method relies on the direct screening of cDNA libraries using pooled single-copy microclones generated by a laser chromosome microdissection in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) procedure. They applied this method to the distal region of human chromosome 4p (4p15-4pter), which contains the Huntington disease (HD) and the Wolf-Hirschhorn syndrome (WHS) loci. Twenty-one nonoverlapping and region-specific cDNA clones encoding novel genes were isolated in this manner. Ten of 21 clones were subregionally assigned tomore » 4p16.1-4pter, and the remainder mapped to the region proximal to 4p16.1. Northern blot and reverse transcription followed by the PCR (RT-PCR) analysis revealed that 16 of these 21 clones detected transcripts in total RNA from human tissues. The method is applicable to other chromosomal regions and is a powerful approach to the isolation of region-specific cDNA clones. 44 refs., 3 figs., 3 tabs.« less

Towards an understanding of British public attitudes concerning human cloning.

PubMed

Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

2007-07-01

The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect public discourse accurately. However, focus group responses did not simply reflect media coverage.

Effects of Cottonwood Leaf Beetle (Coleoptera: Chrysomelidae) Larval Defoliation, Clone, and Season on Populus Foliar Phagostimulants

Treesearch

David R. Coyle; Joel D. McMillin; Richard B. Hall; Elwood R. Hart

2003-01-01

Abstract: The cottonwood leaf beetle, Chrysomela scripta F., is a serious defoliator of plantation Populus in the United States. Current control methods include biorational and synthetic chemicals as well as selecting Populus clones resistant or tolerant to C. scripta...

Inferring Higher Functional Information for RIKEN Mouse Full-Length cDNA Clones With FACTS

PubMed Central

Nagashima, Takeshi; Silva, Diego G.; Petrovsky, Nikolai; Socha, Luis A.; Suzuki, Harukazu; Saito, Rintaro; Kasukawa, Takeya; Kurochkin, Igor V.; Konagaya, Akihiko; Schönbach, Christian

2003-01-01

FACTS (Functional Association/Annotation of cDNA Clones from Text/Sequence Sources) is a semiautomated knowledge discovery and annotation system that integrates molecular function information derived from sequence analysis results (sequence inferred) with functional information extracted from text. Text-inferred information was extracted from keyword-based retrievals of MEDLINE abstracts and by matching of gene or protein names to OMIM, BIND, and DIP database entries. Using FACTS, we found that 47.5% of the 60,770 RIKEN mouse cDNA FANTOM2 clone annotations were informative for text searches. MEDLINE queries yielded molecular interaction-containing sentences for 23.1% of the clones. When disease MeSH and GO terms were matched with retrieved abstracts, 22.7% of clones were associated with potential diseases, and 32.5% with GO identifiers. A significant number (23.5%) of disease MeSH-associated clones were also found to have a hereditary disease association (OMIM Morbidmap). Inferred neoplastic and nervous system disease represented 49.6% and 36.0% of disease MeSH-associated clones, respectively. A comparison of sequence-based GO assignments with informative text-based GO assignments revealed that for 78.2% of clones, identical GO assignments were provided for that clone by either method, whereas for 21.8% of clones, the assignments differed. In contrast, for OMIM assignments, only 28.5% of clones had identical sequence-based and text-based OMIM assignments. Sequence, sentence, and term-based functional associations are included in the FACTS database (http://facts.gsc.riken.go.jp/), which permits results to be annotated and explored through web-accessible keyword and sequence search interfaces. The FACTS database will be a critical tool for investigating the functional complexity of the mouse transcriptome, cDNA-inferred interactome (molecular interactions), and pathome (pathologies). PMID:12819151

Single-step colony assay for screening antibody libraries.

PubMed

Kato, Mieko; Hanyu, Yoshiro

2017-08-10

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10 3 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

Systematic cloning of human minisatellites from ordered array charomid libraries.

PubMed

Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

1990-11-01

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

PubMed

Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

2014-03-01

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants. Copyright © 2013 Elsevier B.V. All rights reserved.

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

PubMed Central

Anderson, Annette Carola; Hellwig, Elmar; Vespermann, Robin; Wittmer, Annette; Schmid, Michael; Karygianni, Lamprini; Al-Ahmad, Ali

2012-01-01

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis. PMID:23152922

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

PubMed

Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

2017-09-11

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Comparison of randomly cloned and whole genomic DNA probes for the detection of Porphyromonas gingivalis and Bacteroides forsythus

PubMed Central

Wong, M.; DiRienzo, J.M.; Lai, C.-H.; Listgarten, M. A.

2012-01-01

Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species. Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 106 target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples. PMID:8636873

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

PubMed Central

Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

2016-01-01

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

Crohns disease with central nervous system vasculitis causing subarachnoid hemorrhage due to aneurysm and cerebral ischemic stroke

PubMed Central

Garge, Shaileshkumar S.; Vyas, Pooja D.; Modi, Pranav D.; Ghatge, Sharad

2014-01-01

Cerebral vasculitis secondary to Crohn's disease (CD) seems to be a very rare phenomenon. We report a 39-year-old male who presented with headache, vomiting, and left-sided weakness in the known case of CD. Cross-sectional imaging (computed tomography and magnetic resonance imaging,) showed right gangliocapsular acute infarct with supraclinoid cistern subarachnoid hemorrhage (SAH). Cerebral digital substraction angiography (DSA) showed dilatation and narrowing of right distal internal carotid artery (ICA). Left ICA was chronically occluded. His inflammatory markers were significantly raised. Imaging features are suggestive of cerebral vasculitis. Arterial and venous infarcts due to thrombosis are known in CD. Our case presented with acute subarachnoid hemorrhage in supraclinoid cistern due to rupture of tiny aneurysm of perforator arteries causing SAH and infarction in right basal ganglia. Patient was treated conservatively with immunosuppression along with medical management of SAH. PMID:25506170

Crohns disease with central nervous system vasculitis causing subarachnoid hemorrhage due to aneurysm and cerebral ischemic stroke.

PubMed

Garge, Shaileshkumar S; Vyas, Pooja D; Modi, Pranav D; Ghatge, Sharad

2014-10-01

Cerebral vasculitis secondary to Crohn's disease (CD) seems to be a very rare phenomenon. We report a 39-year-old male who presented with headache, vomiting, and left-sided weakness in the known case of CD. Cross-sectional imaging (computed tomography and magnetic resonance imaging,) showed right gangliocapsular acute infarct with supraclinoid cistern subarachnoid hemorrhage (SAH). Cerebral digital substraction angiography (DSA) showed dilatation and narrowing of right distal internal carotid artery (ICA). Left ICA was chronically occluded. His inflammatory markers were significantly raised. Imaging features are suggestive of cerebral vasculitis. Arterial and venous infarcts due to thrombosis are known in CD. Our case presented with acute subarachnoid hemorrhage in supraclinoid cistern due to rupture of tiny aneurysm of perforator arteries causing SAH and infarction in right basal ganglia. Patient was treated conservatively with immunosuppression along with medical management of SAH.

[Surgical correction of post-vertebroplasty kyphosis by pedicle substraction osteotomy. Regarding three cases].

PubMed

Bachour, E; Coloma, P; Freitas, E; Messerer, R; Michel, F; Barrey, C

2016-12-01

We report a case of three patients treated with pedicle subtraction osteotomy for post-vertebroplasty kyphosis. These patients were initially treated with a vertebroplasty for vertebral fracture (two cases) and spinal lymphoma (1 case). All of these patients worsened progressively on a clinical and radiographic level with progression of the spinal deformity in the form of kyphosis. The surgery consisted of transpedicular osteotomy instrumented at the level of the vertebra cemented with maximum removal of intra-corporeal cement. One of the three patients required a supplementary anterior approach to achieve good quality bone fusion. In all three cases the post-vertebroplasty kyphosis was able to be reduced by at least 50 % emphasizing the feasibility and relevance of the pedicle subtraction osteotomy in a context of cemented vertebra. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Rocket observations of the ultraviolet airglow during morning twilight

NASA Technical Reports Server (NTRS)

Cebula, R. P.; Feldman, P. D.

1984-01-01

Rocket-borne (Astrobee) UV measurements were made of the terrestrial airglow at morning twilight from 82 and 90 deg zenith angles at altitudes of 90 and 246 km in September 1979. Data were acquired on the NO gamma and delta bands, the 2470 A O II, 1356 A and the 1304 A O I lines, the Lyman-Berge-Hopfield N2 and the Herzberg 02 lines. The zodiacal contribution was substracted to obtain pure airglow data. Spectral analyses supported a larger nighttime decrease of N(4S) than for NO, the latter being in diffusive equilibrium above 190 km altitude. The NO gamma band was directly related to the thermospheric N(4S) contribution, the latter having a density of 2-8 million/cu cm at 200 km. Finally, self-consistent photoionization and photoelectron impact ionization models were derived for the atomic and ionic oxygen emissions.

A unique circovirus-like genome detected in pig feces

USDA-ARS?s Scientific Manuscript database

Using a metagenomic approach and molecular cloning methods, we identified, cloned, and sequenced the complete genome of a novel circular DNA virus, porcine stool-associated virus (PoSCV4), from pig feces. Phylogenetic analysis of the deduced replication initiator protein showed that PoSCV4 is most r...

Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

Quantitative DNA fiber mapping

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich G.

1998-01-01

The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

Inoculation methods for Populus tremuloides resistant to Hypoxylon canker

Treesearch

S. A. Enebak; Michael E. Ostry; N. A. Anderson

1999-01-01

Canker expansion and the amount of callus tissue formed were measured monthly on 60 ramets from each of five trembling aspen (Populus tremuloides Michx.) clones that had been inoculated in wounds with Entoleuca mammata (= Hypoxylon marnmatum (Wahl.) Mill) over a 12-month period. At the clone level, the prevalence...

Novel near-infrared spectrum analysis tool: Synergy adaptive moving window model based on immune clone algorithm.

PubMed

Wang, Shenghao; Zhang, Yuyan; Cao, Fuyi; Pei, Zhenying; Gao, Xuewei; Zhang, Xu; Zhao, Yong

2018-02-13

This paper presents a novel spectrum analysis tool named synergy adaptive moving window modeling based on immune clone algorithm (SA-MWM-ICA) considering the tedious and inconvenient labor involved in the selection of pre-processing methods and spectral variables by prior experience. In this work, immune clone algorithm is first introduced into the spectrum analysis field as a new optimization strategy, covering the shortage of the relative traditional methods. Based on the working principle of the human immune system, the performance of the quantitative model is regarded as antigen, and a special vector corresponding to the above mentioned antigen is regarded as antibody. The antibody contains a pre-processing method optimization region which is created by 11 decimal digits, and a spectrum variable optimization region which is formed by some moving windows with changeable width and position. A set of original antibodies are created by modeling with this algorithm. After calculating the affinity of these antibodies, those with high affinity will be selected to clone. The regulation for cloning is that the higher the affinity, the more copies will be. In the next step, another import operation named hyper-mutation is applied to the antibodies after cloning. Moreover, the regulation for hyper-mutation is that the lower the affinity, the more possibility will be. Several antibodies with high affinity will be created on the basis of these steps. Groups of simulated dataset, gasoline near-infrared spectra dataset, and soil near-infrared spectra dataset are employed to verify and illustrate the performance of SA-MWM-ICA. Analysis results show that the performance of the quantitative models adopted by SA-MWM-ICA are better especially for structures with relatively complex spectra than traditional models such as partial least squares (PLS), moving window PLS (MWPLS), genetic algorithm PLS (GAPLS), and pretreatment method classification and adjustable parameter changeable size moving window PLS (CA-CSMWPLS). The selected pre-processing methods and spectrum variables are easily explained. The proposed method will converge in few generations and can be used not only for near-infrared spectroscopy analysis but also for other similar spectral analysis, such as infrared spectroscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Somatic Cell Nuclear Transfer in the Mouse

NASA Astrophysics Data System (ADS)

Kishigami, Satoshi; Wakayama, Teruhiko

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

Production of cloned mice by somatic cell nuclear transfer.

PubMed

Kishigami, Satoshi; Wakayama, Sayaka; Thuan, Nguyen Van; Ohta, Hiroshi; Mizutani, Eiji; Hikichi, Takafusa; Bui, Hong-Thuy; Balbach, Sebastian; Ogura, Atsuo; Boiani, Michele; Wakayama, Teruhiko

2006-01-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains < 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell's genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in > or = 3 months.

Using somatic-cell nuclear transfer to study aging.

PubMed

Kishigami, Satoshi; Lee, Ah Reum; Wakayama, Teruhiko

2013-01-01

In mammals, a diploid genome following fertilization of haploid cells, an egg, and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual's inevitable demise. Since it was first reported in 1997 that Dolly the sheep had been cloned, many mammalian species have been cloned successfully using somatic-cell nuclear transfer (SCNT). The success of SCNT in mammals enables us not only to reproduce offspring without germ cells, that is, to "passage" a unique diploid genome, but also to address valuable biological questions on development, nuclear reprogramming, and epigenetic memory. Successful cloning can also support epigenetic reprogramming where the aging clock is reset or reversed. Recent work using iPS cell technology has explored the practicality and led to the recapitulation of premature aging with iPSCs from progeroid laminopathies. As a result, reprogramming tools are also expected to contribute to studying biological age. However, the efficiency of animal cloning is still low in most cases and the mechanism of reprogramming in cloned embryos is still largely unclear. Here, based on recent advances, we describe an improved, more efficient mouse cloning protocol using histone deacetylase inhibitors (HDACis) and latrunculin A, which increases the success rates of producing cloned mice or establishing ES cells fivefold. This improved method of cloning will provide a strong tool to address many issues including biological aging more easily and with lower cost.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cernoch, Antonin; Soubusta, Jan; Celechovska, Lucie

We report on experimental implementation of the optimal universal asymmetric 1->2 quantum cloning machine for qubits encoded into polarization states of single photons. Our linear-optical machine performs asymmetric cloning by partially symmetrizing the input polarization state of signal photon and a blank copy idler photon prepared in a maximally mixed state. We show that the employed method of measurement of mean clone fidelities exhibits strong resilience to imperfect calibration of the relative efficiencies of single-photon detectors used in the experiment. Reliable characterization of the quantum cloner is thus possible even when precise detector calibration is difficult to achieve.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Two level pedicle substraction osteotomies for the treatment of severe fixed sagittal plane deformity: computer software-assisted preoperative planning and assessing.

PubMed

Atici, Yunus; Akman, Yunus Emre; Balioglu, Mehmet Bulent; Kargin, Deniz; Kaygusuz, Mehmet Akif

2016-08-01

To evaluate the efficacy of two level pedicle substraction osteotomies (PSOs) planned preoperatively with a computer software, in the patients with severe fixed sagittal plane deformities. In the literature, there are studies indicating that two level PSOs may be required in severe cases. However, the results of two level PSOs preoperatively planned with computer software-assistance have not yet been reported in the English literature. Severe fixed sagittal plane deformities of 11 patients are described. Preoperative surgical planning was done with the aid of a computer software. Two level PSOs were indicated after the process. After the application of the indicated surgical technique, clinical and radiological results were evaluated in the preoperative, the early postoperative periods and during the last follow-up. The mean sagittal vertical axis was found as 190.5 (range 161-220) mm in the preoperative period, 23.5 (range -27 to 61) mm in the early postoperative period (P < 0.001) (87.7 % correction) and 34.5 (range -3 to 55) mm during the last follow-up (P < 0.001). The mean pelvic tilt (PT) significantly decreased from 38.3° (range 21°-63°) preoperatively to 23.8° (range 18°-42°) postoperatively (P = 0.008) and to 27.5° (range 17°-42°) during the last follow-up (P = 0.042). The mean lumbar lordosis (LL) was 2.8° (range -29° to 20°) preoperatively, -35.6° (range -54° to 23°) early postoperatively (P < 0.001) and -33.6° (range -52° to 20°) during the last follow-up (P < 0.001). The average amount of bleeding was 5345 (range 2600-7415) ml. Although a statistically significant correction was obtained, the mean PT and PI-LL value could not be restored in physiological limits during the last follow-up. Thus, two level PSOs performed after computer software (surgimap) assisted preoperative planning failed to correct severe fixed sagittal plane deformities. Besides, this procedure is of possible risks for major complications such as a result of excessive bleeding. We recommend that two level PSOs should be rarely indicated, but preferred as an alternative technique only in the most severe cases.

Bacterial biodiversity from an anaerobic up flow bioreactor with ANAMMOX activity inoculated with swine sludge

USDA-ARS?s Scientific Manuscript database

The present study aimed to identify organisms with ANAMMOX activity in a reactor maintained in a laboratory. Molecular methods as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) and cloning of 16S-rDNA genes probing for Planctomycetes were performed. Seventeen clones were ...

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

USDA-ARS?s Scientific Manuscript database

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

DNA book.

PubMed

Kawai, Jun; Hayashizaki, Yoshihide

2003-06-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%-100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition.

Genomic validation of PB 260 clone of rubber (Hevea brasiliensis) at Cikumpay Plantation by SSR marker

NASA Astrophysics Data System (ADS)

Royani, J. I.; Safarrida, A.; Rachmawati, I.; Khairiyah, H.; Mustika, I. P.; Suyono, A.; Rudiyana, Y.; Kubil; Nurjaya; Arianto, A.

2017-05-01

Rubber from Hevea brasiliensis is the only commercial natural rubber in the world. Propagation of rubber trees usually done by grafting and seed germination. BPPT had been producing rubber tree by in vitro technique with embryo somatic methods. Validation of mother plant for in vitro propagation is important to compare between mother plant and propagated plants. The aim for this research was to validation of PB 260 clone that planted at Cikumpay Plantation by SSR marker. Sampling of 10 rubber leaves were done at Cikumpay Plantation based on GPS position from the area of PB 260 clone. Rubber leaves were isolated with CTAB modification method to obtained DNA. Four of SSR primers from rubber, i.e.: hmac 4, hmac 5, hmct 1, and hmct 5, were used as primers to amplification of rubber DNA. The result showed that no band that different from 10 rubber of PB 260 clone at Cikumpay Plantation. This research will continue to compare genomic validation between mother plant and propagated plants that had been produced from BPPT.

Bacterial community analysis of swine manure treated with autothermal thermophilic aerobic digestion.

PubMed

Han, Il; Congeevaram, Shankar; Ki, Dong-Won; Oh, Byoung-Taek; Park, Joonhong

2011-02-01

Due to the environmental problems associated with disposal of livestock sludge, many stabilization studies emphasizing on the sludge volume reduction were performed. However, little is known about the microbial risk present in sludge and its stabilized products. This study microbiologically explored the effects of anaerobic lagoon fermentation (ALF) and autothermal thermophilic aerobic digestion (ATAD) on pathogen-related risk of raw swine manure by using culture-independent 16S rDNA cloning and sequencing methods. In raw swine manure, clones closely related to pathogens such as Dialister pneumosintes, Erysipelothrix rhusiopathiae, Succinivibrioan dextrinosolvens, and Schineria sp. were detected. Meanwhile, in the mesophilic ALF-treated swine manure, bacterial community clones closely related to pathogens such as Schineria sp. and Succinivibrio dextrinosolvens were still detected. Interestingly, the ATAD treatment resulted in no detection of clones closely related to pathogens in the stabilized thermophilic bacterial community, with the predominance of novel Clostridia class populations. These findings support the superiority of ATAD in selectively reducing potential human and animal pathogens compared to ALF, which is a typical manure stabilization method used in livestock farms.

Nuclear transfer to prevent mitochondrial DNA disorders: revisiting the debate on reproductive cloning.

PubMed

Bredenoord, A L; Dondorp, W; Pennings, G; De Wert, G

2011-02-01

Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Emotional reactions to human reproductive cloning.

PubMed

May, Joshua

2016-01-01

Extant surveys of people's attitudes towards human reproductive cloning focus on moral judgements alone, not emotional reactions or sentiments. This is especially important given that some (especially Leon Kass) have argued against such cloning on the ground that it engenders widespread negative emotions, like disgust, that provide a moral guide. To provide some data on emotional reactions to human cloning, with a focus on repugnance, given its prominence in the literature. This brief mixed-method study measures the self-reported attitudes and emotions (positive or negative) towards cloning from a sample of participants in the USA. Most participants condemned cloning as immoral and said it should be illegal. The most commonly reported positive sentiment was by far interest/curiosity. Negative emotions were much more varied, but anxiety was the most common. Only about a third of participants selected disgust or repugnance as something they felt, and an even smaller portion had this emotion come to mind prior to seeing a list of options. Participants felt primarily interested and anxious about human reproductive cloning. They did not primarily feel disgust or repugnance. This provides initial empirical evidence that such a reaction is not appropriately widespread. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Vectors for co-expression of an unrestricted number of proteins

PubMed Central

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-01-01

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

PubMed Central

Li, Yunlong; Zhang, Yong; Lu, Pei; Rayner, Simon; Chen, Shiyun

2012-01-01

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions. PMID:23185557

How to improve the success rate of mouse cloning technology.

PubMed

Thuan, Nguyen Van; Kishigami, Satoshi; Wakayama, Teruhiko

2010-02-01

It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

PubMed Central

Benoit, Roger M.; Ostermeier, Christian; Geiser, Martin; Li, Julia Su Zhou; Widmer, Hans; Auer, Manfred

2016-01-01

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides. PMID:27073895

A hybrid approach identifies metabolic signatures of high-producers for chinese hamster ovary clone selection and process optimization.

PubMed

Popp, Oliver; Müller, Dirk; Didzus, Katharina; Paul, Wolfgang; Lipsmeier, Florian; Kirchner, Florian; Niklas, Jens; Mauch, Klaus; Beaucamp, Nicola

2016-09-01

In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. This requires applying appropriate methods during bioprocess development to enable meaningful characterization of CHO clones and processes. Here, we present a novel hybrid approach for supporting comprehensive characterization of metabolic clone performance. The approach combines metabolite profiling with multivariate data analysis and fluxomics to enable a data-driven mechanistic analysis of key metabolic traits associated with desired cell phenotypes. We applied the methodology to quantify and compare metabolic performance in a set of 10 recombinant CHO-K1 producer clones and a host cell line. The comprehensive characterization enabled us to derive an extended set of clone performance criteria that not only captured growth and product formation, but also incorporated information on intracellular clone physiology and on metabolic changes during the process. These criteria served to establish a quantitative clone ranking and allowed us to identify metabolic differences between high-producing CHO-K1 clones yielding comparably high product titers. Through multivariate data analysis of the combined metabolite and flux data we uncovered common metabolic traits characteristic of high-producer clones in the screening setup. This included high intracellular rates of glutamine synthesis, low cysteine uptake, reduced excretion of aspartate and glutamate, and low intracellular degradation rates of branched-chain amino acids and of histidine. Finally, the above approach was integrated into a workflow that enables standardized high-content selection of CHO producer clones in a high-throughput fashion. In conclusion, the combination of quantitative metabolite profiling, multivariate data analysis, and mechanistic network model simulations can identify metabolic traits characteristic of high-performance clones and enables informed decisions on which clones provide a good match for a particular process platform. The proposed approach also provides a mechanistic link between observed clone phenotype, process setup, and feeding regimes, and thereby offers concrete starting points for subsequent process optimization. Biotechnol. Bioeng. 2016;113: 2005-2019. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

PubMed

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

The effect of sampling techniques used in the multiconfigurational Ehrenfest method

NASA Astrophysics Data System (ADS)

Symonds, C.; Kattirtzi, J. A.; Shalashilin, D. V.

2018-05-01

In this paper, we compare and contrast basis set sampling techniques recently developed for use in the ab initio multiple cloning method, a direct dynamics extension to the multiconfigurational Ehrenfest approach, used recently for the quantum simulation of ultrafast photochemistry. We demonstrate that simultaneous use of basis set cloning and basis function trains can produce results which are converged to the exact quantum result. To demonstrate this, we employ these sampling methods in simulations of quantum dynamics in the spin boson model with a broad range of parameters and compare the results to accurate benchmarks.

The effect of sampling techniques used in the multiconfigurational Ehrenfest method.

PubMed

Symonds, C; Kattirtzi, J A; Shalashilin, D V

2018-05-14

In this paper, we compare and contrast basis set sampling techniques recently developed for use in the ab initio multiple cloning method, a direct dynamics extension to the multiconfigurational Ehrenfest approach, used recently for the quantum simulation of ultrafast photochemistry. We demonstrate that simultaneous use of basis set cloning and basis function trains can produce results which are converged to the exact quantum result. To demonstrate this, we employ these sampling methods in simulations of quantum dynamics in the spin boson model with a broad range of parameters and compare the results to accurate benchmarks.

Bacterial diversity associated with the rotifer Brachionus plicatilis sp. complex determined by culture-dependent and -independent methods.

PubMed

Ishino, Ryota; Iehata, Shunpei; Nakano, Miyo; Tanaka, Reiji; Yoshimatsu, Takao; Maeda, Hiroto

2012-03-01

The bacterial communities associated with rotifers (Brachionus plicatilis sp. complex) and their culture water were determined using culture-dependent and -independent methods (16S rRNA gene clone library). The bacterial communities determined by the culture-independent method were more diverse than those determined by the culture-dependent method. Although the culture-dependent method indicated the bacterial community of rotifers was relatively similar to that of the culture water, 16S rRNA gene clone library analyses revealed a great difference between the two microbiotas. Our results suggest that most bacteria associated with rotifers are not easily cultured using conventional methods, and that the microbiota of rotifers do not correspond with that of the culture water completely.

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future.

PubMed

Mameli, M

2007-02-01

Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. In this paper, two versions of this view are discussed. According to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. According to the second version, these biotechnologies would undermine autonomy by violating these people's right to an open future. There is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans.

Reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future

PubMed Central

Mameli, M

2007-01-01

Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. In this paper, two versions of this view are discussed. According to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. According to the second version, these biotechnologies would undermine autonomy by violating these people's right to an open future. There is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans. PMID:17264194

Birth of cloned mice from vaginal smear cells after somatic cell nuclear transfer.

PubMed

Kuwayama, Hiroki; Tanabe, Yoshiaki; Wakayama, Teruhiko; Kishigami, Satoshi

2017-05-01

Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic-cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasibility of these cells as sources of nuclei for somatic-cell cloning using nuclear transfer. Estrous cycles generally comprise proestrus, estrus, metestrus, and diestrus stages. In the present experiments, more than 60% of cells were nucleated in vaginal smears from all but the estrus stage. However, after somatic-cell nuclear transfer of cells from proestrus, metestrus, and diestrus stages, 66%, 50%, and 72% of cloned embryos developed to the morula/blastocyst, and cloned female mouse birth rates after embryo transfer were 1.5%, 0.3%, and 1%, respectively. These results show that noninvasively collected vaginal smears contain somatic cells that can be used to clone female mice. Copyright © 2017. Published by Elsevier Inc.

Influence of embryo handling and transfer method on pig cloning efficiency.

PubMed

Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

2015-03-01

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

Preimplantation development of somatic cell cloned embryos in the common marmoset (Callithrix jacchus).

PubMed

Sotomaru, Yusuke; Hirakawa, Reiko; Shimada, Akiko; Shiozawa, Seiji; Sugawara, Ayako; Oiwa, Ryo; Nobukiyo, Asako; Okano, Hideyuki; Tamaoki, Norikazu; Nomura, Tatsuji; Hiyama, Eiso; Sasaki, Erika

2009-12-01

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

PubMed

Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

2016-02-01

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-10-20

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-11-03

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

Sequence Typing Confirms that a Predominant Listeria monocytogenes Clone Caused Human Listeriosis Cases and Outbreaks in Canada from 1988 to 2010

PubMed Central

Reimer, Aleisha; Verghese, Bindhu; Lok, Mei; Ziegler, Jennifer; Farber, Jeffrey; Pagotto, Franco; Graham, Morag; Nadon, Celine A.

2012-01-01

Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades. PMID:22337989

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1986-11-26

cloning at the SalI site of pUCI8 vector DNA, iii) by treatment with EcoRl DNA methylase, ligation to EcoRI and cloning at the EcoRl site of pUCI8...cDNA to synthetic Sail linker 10 2.3.10 Treatment of DEN-2 cDNA with EcoRi methylase, followed 10 by ligation to EcoRI linkers and digestion with...picked by the mini plasmid preparation method as described in Maniatis et al. (1982). The procedure followed involved briefly treatment with a

Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning.

PubMed

Kurome, Mayuko; Fujimura, Tatsuya; Murakami, Hiroshi; Takahagi, Yoichi; Wako, Naohiro; Ochiai, Takashi; Miyazaki, Koji; Nagashima, Hiroshi

2003-01-01

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.

Asymmetric single-strand polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants

USDA-ARS?s Scientific Manuscript database

We needed to obtain an alternative to conventional cloning to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies in potato, to save time and money and to avoid problems typically encountered in cloning. We tested a variety of SSCP protocols to include pu...

Female reproductive success decreases with display size in monkshood, Aconitum kusnezoffii (Ranunculaceae)

PubMed Central

Liao, Wan-Jin; Hu, Yi; Zhu, Bi-Ru; Zhao, Xia-Qing; Zeng, Yan-Fei; Zhang, Da-Yong

2009-01-01

Background and Aims Reduction in female fitness in large clones can occur as a result of increased geitonogamous self-fertilization and its influence through inbreeding depression. This possibility was investigated in the self-compatible, bee-pollinated perennial herb Aconitum kusnezoffii which varies in clone size. Methods Field investigations were conducted on pollinator behaviour, flowering phenology and variation in seed set. The effects of self-pollination following controlled self- and cross-pollination were also examined. Selfing rates of differently sized clones were assessed using allozyme markers. Key Results High rates of geitonogamous pollination were associated with large display size. Female fitness at the ramet level decreased with clone size. Fruit and seed set under cross-pollination were significantly higher than those under self-pollination. The pre-dispersal inbreeding depression was estimated as 0·502 based on the difference in seed set per flower between self- and cross-pollinated flowers. Selfing rates of differently sized clones did not differ. Conclusions It is concluded that in A. kusnezoffii the negative effects of self-pollination causing reduced female fertility with clone size arise primarily from a strong early-acting inbreeding depression leading to the abortion of selfed embryos prior to seed maturation. PMID:19767308

The potential and biological test on cloned cassava crop remains on local sheep

NASA Astrophysics Data System (ADS)

Ginting, R.; Umar, S.; Hanum, C.

2018-02-01

This research aims at knowing the potential of cloned cassava crop remains dry matter and the impact of the feeding of the cloned cassava crop remains based complete feed on the consumption, the body weight gain, and the feed conversion of the local male sheep with the average of initial body weight of 7.75±1.75 kg. The design applied in the first stage research was random sampling method with two frames of tile and the second stage research applied Completely Randomized Design (CRD) with three (3) treatments and four (4) replicates. These treatments consisted of P1 (100% grass); P2 (50% grass, 50% complete feed pellet); P3 (100% complete feed from the raw material of cloned cassava crop remaining). Statistical tests showed that the feeding of complete feed whose raw material was from cloned cassava crop remains gave a highly significant impact on decreasing feed consumption, increasing body weight, lowering feed conversion, and increasing crude protein digestibility. The conclusion is that the cloned cassava crop remains can be used as complete sheep feed to replace green grass and can give the best result.

Shifts in the Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010

PubMed Central

Boswihi, Samar S.; Udo, Edet E.; Al-Sweih, Noura

2016-01-01

Background As the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is constantly changing globally, determining the prevailing MRSA clones in a local healthcare facility is important for better management of infections. This study investigated clonal composition and distribution of MRSA isolates in Kuwait’s hospitals using a combination of molecular typing methods. Materials and Methods In total, 400 non-repeat MRSA isolates were obtained between 1992 and 2010 in 13 public hospitals and were characterized using antibiogram, SCCmec typing, spa typing, and multilocus-sequence typing. Clonal assignment and detection of virulence factors and antibiotic resistance genes were performed by DNA microarray. Results The isolates were resistant to kanamycin (74.2%), erythromycin (69.5%), tetracycline (66.7%), gentamicin (61%), ciprofloxacin, (61%), fusidic acid (53.5%), clindamycin (41.5%), high-level mupirocin resistance (5.2%) and carried aphA3, aacA-aphD, ermA, ermC, mupA, tetK, tetM, fusC and far1. Molecular typing revealed 31 different MRSA clones consisting of ST239-MRSA-III (52.2%), ST22-MRSA-IV (9.2%), ST80-MRSA-IV (7.5%), ST5-MRSA-II/IV/V/VI (6.5%), ST30-MRSA-IV (3.5%), ST241-MRSA-III (2.7%), ST6-MRSA-IV (2.2%), ST36-MRSA-II (2%) and ST772-MRSA-V (1.75%). The isolates differed in the carriage of genes for enterotoxins, Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (tst-1), arginine catabolic mobile element (ACME) and exfoliative toxins. The number of clones increased from one (ST239-III-t037) in 1992 to 30 in 2010 including ST8-IV-t008 [PVL+] [ACME+] (USA300), ST772-V (Bengal Bay clone) and ST2816 identified for the first time in Kuwait. Conclusion The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime. Although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime, the newly emerged clones consisted mostly of community-associated. PMID:27631623

Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.

PubMed

Kojima, T; Habu, Y; Iida, S; Ogihara, Y

2000-05-01

The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

PubMed

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potential candidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research. Creative Commons Attribution License

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

PubMed Central

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

PubMed

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L

2014-01-01

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 μM and 12.2 ± 2.1μM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

Production of cloned mice from somatic cells, ES cells, and frozen bodies.

PubMed

Wakayama, Sayaka; Mizutani, Eiji; Wakayama, Teruhiko

2010-01-01

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach" and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

PubMed Central

Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

2009-01-01

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

Capturing diversity of marine heterotrophic protists: one cell at a time

PubMed Central

Heywood, Jane L; Sieracki, Michael E; Bellows, Wendy; Poulton, Nicole J; Stepanauskas, Ramunas

2011-01-01

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups. PMID:20962875

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

PubMed Central

2012-01-01

Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS. PMID:22480370

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

PubMed

Inoue, Kimiko; Kohda, Takashi; Sugimoto, Michihiko; Sado, Takashi; Ogonuki, Narumi; Matoba, Shogo; Shiura, Hirosuke; Ikeda, Rieko; Mochida, Keiji; Fujii, Takashi; Sawai, Ken; Otte, Arie P; Tian, X Cindy; Yang, Xiangzhong; Ishino, Fumitoshi; Abe, Kuniya; Ogura, Atsuo

2010-10-22

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

PubMed

Bujaki, Erika

2016-01-01

The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

Vascular alterations underlie developmental problems manifested in cloned cattle before or after birth.

PubMed

Maiorka, Paulo Cesar; Favaron, Phelipe Oliveira; Mess, Andrea Maria; dos Santos, Caio Rodrigues; Alberto, Miryan Lanca; Meirelles, Flavio Vieira; Miglino, Maria Angelica

2015-01-01

Although assisted reproductive techniques are commonly applied in humans and animals, they are frequently associated with major developmental deficits and reduced viability. To explore abnormalities associated with cloning or nuclear transfer (NT) as the most invasive of these methods, we used a bovine model to characterize abnormalities. Detailed necropsy examinations were done on 13 calves that died soon after birth; in addition, we included data from embryos and fetuses (produced by NT) that terminated prematurely. Bovine clones that survived until the neonatal period differed quantitatively and qualitatively from in-vivo-derived cattle. Although alterations affected a variety of organs (e.g. heart, lung and liver), there was a clear association with abberant vascular developmental during the early intrauterine phase. Therefore, we concluded that vascular problems were key alterations induced by cloning (presumably via epigenetic modifications).

Vascular Alterations Underlie Developmental Problems Manifested in Cloned Cattle before or after Birth

PubMed Central

Favaron, Phelipe Oliveira; dos Santos, Caio Rodrigues; Alberto, Miryan Lanca; Meirelles, Flavio Vieira; Miglino, Maria Angelica

2015-01-01

Although assisted reproductive techniques are commonly applied in humans and animals, they are frequently associated with major developmental deficits and reduced viability. To explore abnormalities associated with cloning or nuclear transfer (NT) as the most invasive of these methods, we used a bovine model to characterize abnormalities. Detailed necropsy examinations were done on 13 calves that died soon after birth; in addition, we included data from embryos and fetuses (produced by NT) that terminated prematurely. Bovine clones that survived until the neonatal period differed quantitatively and qualitatively from in-vivo-derived cattle. Although alterations affected a variety of organs (e.g. heart, lung and liver), there was a clear association with abberant vascular developmental during the early intrauterine phase. Therefore, we concluded that vascular problems were key alterations induced by cloning (presumably via epigenetic modifications). PMID:25584533

Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses

USDA-ARS?s Scientific Manuscript database

This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). A full-length C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog...

DNA Book

PubMed Central

Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%–100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition. PMID:12819147

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

PubMed Central

Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

2013-01-01

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. PMID:23667668

Effects of trichostatin A on In vitro development and DNA methylation level of the satellite I region of swamp buffalo (Bubalus bubalis) cloned embryos.

PubMed

Srirattana, Kanokwan; Ketudat-Cairns, Mariena; Nagai, Takashi; Kaneda, Masahiro; Parnpai, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82-85%), cleavage (79-84%) and development to the 8-cell stage (59-65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.

Effects of Trichostatin A on In Vitro Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (Bubalus bubalis) Cloned Embryos

PubMed Central

SRIRATTANA, Kanokwan; KETUDAT-CAIRNS, Mariena; NAGAI, Takashi; KANEDA, Masahiro; PARNPAI, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed. PMID:24909601

pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.

PubMed

Campbell, A Malcolm; Eckdahl, Todd; Cronk, Brian; Andresen, Corinne; Frederick, Paul; Huckuntod, Samantha; Shinneman, Claire; Wacker, Annie; Yuan, Jason

2014-01-01

The Vision and Change report recommended genuine research experiences for undergraduate biology students. Authentic research improves science education, increases the number of scientifically literate citizens, and encourages students to pursue research. Synthetic biology is well suited for undergraduate research and is a growing area of science. We developed a laboratory module called pClone that empowers students to use advances in molecular cloning methods to discover new promoters for use by synthetic biologists. Our educational goals are consistent with Vision and Change and emphasize core concepts and competencies. pClone is a family of three plasmids that students use to clone a new transcriptional promoter or mutate a canonical promoter and measure promoter activity in Escherichia coli. We also developed the Registry of Functional Promoters, an open-access database of student promoter research results. Using pre- and posttests, we measured significant learning gains among students using pClone in introductory biology and genetics classes. Student posttest scores were significantly better than scores of students who did not use pClone. pClone is an easy and affordable mechanism for large-enrollment labs to meet the high standards of Vision and Change. © 2014 A. M. Campbell et al. CBE—Life Sciences Education © 2014 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Case history development of a hybrid poplar nursery at Reynolds Metals Company, Massena, NY. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marler, R.L.

1983-03-01

The methods used to establish the hybrid poplar nursery and the results obtained in the first year's operation are discussed. It also documents the history of the project. Hybrid poplar clones were established in the nursery in 1980 and their growth performance was evaluated after one growing season. The results were most promising. The mean survivability rate ranged from 84.92% to 90.58% per clone, while the clone height growth ranged from 58.16 inches to 76.47 inches over the first growing season. Approximately 71,000 cuttings were estimated to be available for outplanting during the Spring of 1982.

Science and technology of farm animal cloning: state of the art.

PubMed

Vajta, Gábor; Gjerris, Mickey

2006-05-01

Details of the first mammal born after nuclear transfer cloning were published by Steen Malte Willadsen in 1986. In spite of its enormous scientific significance, this discovery failed to trigger much public concern, possibly because the donor cells were derived from pre-implantation stage embryos. The major breakthrough in terms of public recognition has happened when Ian Wilmut et al. [Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable offspring derived from fetal és adult mammalian cells. Nature 385, 810-813] described the successful application of almost exactly the same method, but using the nuclei of somatic cells from an adult mammal, to create Dolly the sheep. It has become theoretically possible to produce an unlimited number of genetic replicates from an adult animal or a post-implantation foetus. Since 1997 a number of different species including pigs, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research and the biomedical sector. The next step seems to be to implement cloning in the agricultural production system and several animals have been developed in this direction. This article reviews the current state of the art of farm animal cloning from a scientific and technological perspective, describes the animal welfare problems and critically assess different applications of farm animal cloning. The scope is confined to animal biotechnologies in which the use of cell nuclear transfer is an essential part and extends to both biomedical and agricultural applications of farm animal cloning. These applications include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available; instead we pinpoint issues and events pivotal to the development of current farm animal cloning practices and their possible applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Meng-Zheng; School of Physics and Electronic Information, Huaibei Normal University, Huaibei 235000; Ye, Liu, E-mail: yeliu@ahu.edu.cn

An efficient scheme is proposed to implement phase-covariant quantum cloning by using a superconducting transmon qubit coupled to a microwave cavity resonator in the strong dispersive limit of circuit quantum electrodynamics (QED). By solving the master equation numerically, we plot the Wigner function and Poisson distribution of the cavity mode after each operation in the cloning transformation sequence according to two logic circuits proposed. The visualizations of the quasi-probability distribution in phase-space for the cavity mode and the occupation probability distribution in the Fock basis enable us to penetrate the evolution process of cavity mode during the phase-covariant cloning (PCC)more » transformation. With the help of numerical simulation method, we find out that the present cloning machine is not the isotropic model because its output fidelity depends on the polar angle and the azimuthal angle of the initial input state on the Bloch sphere. The fidelity for the actual output clone of the present scheme is slightly smaller than one in the theoretical case. The simulation results are consistent with the theoretical ones. This further corroborates our scheme based on circuit QED can implement efficiently PCC transformation.« less

Identification of downy mildew resistance gene candidates by positional cloning in maize (Zea mays subsp. mays; Poaceae)1

PubMed Central

Kim, Jae Yoon; Moon, Jun-Cheol; Kim, Hyo Chul; Shin, Seungho; Song, Kitae; Kim, Kyung-Hee; Lee, Byung-Moo

2017-01-01

Premise of the study: Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. Methods and Results: Downy mildew (DM)–resistant maize was selected from five cultivars using a spreader row technique. Positional cloning and bioinformatics tools were used to identify the DM-resistance quantitative trait locus marker (bnlg1702) and 47 protein-coding gene annotations. Eventually, five DM-resistance gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative reverse-transcription PCR (RT-PCR) without fine mapping of the bnlg1702 locus. Conclusions: The combined protocol with the spreader row technique, quantitative trait locus positional cloning, and quantitative RT-PCR was effective for identifying DM-resistance candidate genes. This cloning approach may be applied to other whole-genome-sequenced crops or resistance to other diseases. PMID:28224059

Alignment-Independent Comparisons of Human Gastrointestinal Tract Microbial Communities in a Multidimensional 16S rRNA Gene Evolutionary Space▿

PubMed Central

Rudi, Knut; Zimonja, Monika; Kvenshagen, Bente; Rugtveit, Jarle; Midtvedt, Tore; Eggesbø, Merete

2007-01-01

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities. PMID:17337554

Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate.

PubMed

Bowei, Chen; Xingyu, Liu; Wenyan, Liu; Jiankang, Wen

2009-11-01

The microbial communities of leachate from a bioleaching heap located in China were analyzed using the 16S rRNA gene clone library and real-time quantitative PCR. Both methods showed that Leptospirillum spp. were the dominant bacteria, and Ferroplasma acidiphilum were the only archaea detected in the leachate. Clone library results indicated that nine operational taxonomic units (OTUs) were obtained, which fell into four divisions, the Nitrospirae (74%), the gamma-Proteobacteria (14%), the Actinobacteria (6%) and the Euryarchaeota (6%). The results obtained by real-time PCR in some ways were the same as clone library analysis. Furthermore, Sulfobacillus spp., detected only by real-time PCR, suggests that real-time PCR was a reliable technology to study the microbial communities in bioleaching environments. It is a useful tool to assist clone library analysis, to further understand microbial consortia and to have comprehensive and exact microbiological information about bioleaching environments. Finally, the interactions among the microorganisms detected in the leachate were summarized according to the characteristics of these species.

An investigative graduate laboratory course for teaching modern DNA techniques.

PubMed

de Lencastre, Alexandre; Thomas Torello, A; Keller, Lani C

2017-07-08

This graduate-level DNA methods laboratory course is designed to model a discovery-based research project and engages students in both traditional DNA analysis methods and modern recombinant DNA cloning techniques. In the first part of the course, students clone the Drosophila ortholog of a human disease gene of their choosing using Gateway ® cloning. In the second part of the course, students examine the expression of their gene of interest in human cell lines by reverse transcription PCR and learn how to analyze data from quantitative reverse transcription PCR (qRT-PCR) experiments. The adaptability of the Gateway ® cloning system is ideally suited for students to design and create different types of expression constructs to achieve a particular experimental goal (e.g., protein purification, expression in cell culture, and/or subcellular localization), and the genes chosen can be aligned to the research interests of the instructor and/or ongoing research in a department. Student evaluations indicate that the course fostered a genuine excitement for research and in depth knowledge of both the techniques performed and the theory behind them. Our long-term goal is to incorporate this DNA methods laboratory as the foundation for an integrated laboratory sequence for the Master of Science degree program in Molecular and Cellular Biology at Quinnipiac University, where students use the reagents and concepts they developed in this course in subsequent laboratory courses, including a protein methods and cell culture laboratory. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):351-359, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

[Mystery and problems of cloning].

PubMed

Nikitin, V A

2010-01-01

The attention of investigators is attracted to the fact that, in spite of great efforts in mammalian cloning, advances that have been made in this area of research are not great, and cloned animals have developmental pathologies often incompatible with life and/or reproduction ability. It is yet not clear what technical or biological factors underlie this, and how they are connected or interact with each other, which is more realistic strategically. There is a great number of articles dealing with the influence of cloning with the nuclear transfer on genetic and epigenetic reprogramming of donor cells. At the same time we can see the practical absence of analytical investigations concerning the technology of cloning as such, its weak points, and possible sources of cellular trauma in the course of microsurgery of nuclear transfer or twinning. This article discusses step by step several nuclear transfer techniques and the methods of dividing early preimplanted embryos for twinning with the aim to reveal possible sources of cell damage during micromanipulation that may have negative influence on the development of cloned organisms. Several new author's technologies based on the study of cell biophysical characteristics are described, which allow one to avoid cellular trauma during manipulation and minimize the possibility of cell damage at any rate.

Variations and voids: the regulation of human cloning around the world

PubMed Central

Pattinson, Shaun D; Caulfield, Timothy

2004-01-01

Background No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Methods Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC). Results While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. Conclusions There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain. PMID:15596013

Biocompatibility studies of natural rubber latex from different tree clones and collection methods.

PubMed

Floriano, Juliana Ferreira; da Mota, Lígia Souza Lima Silveira; Furtado, Edson Luiz; Rossetto, Victor José Vieira; Graeff, Carlos F O

2014-02-01

Natural rubber latex (NRL) has several features that make it an excellent biomaterial to promote the growth and repair of tissues, skin and bones. Most of the research with NRL membranes uses a mixture of different clones and chemical preservatives in the collection process. In this study, we compared five clones that produce NRL, seeking to identify their differences in biocompatibility. The clones studied were RRIM 600, PB 235, GT1, PR 255 and IAN 873 commonly found in plantations in Brazil. We did also study the effect of ammonia used during latex collection. NRL membranes were prepared aseptically and sterilized. In the in vitro tests, the membranes remained in direct contact with mouse fibroblasts cells for three periods, 24, 48 and 72 h. In the in vivo tests, the membranes were implanted subcutaneously in rabbits. The results indicated the biocompatibility of the membranes obtained from all clones. Membranes from the clones RRIM 600 and IAN 873 induced greater cell proliferation, suggesting greater bioactivity. It was found that the membranes made from latex that was in contact with ammonia during collection, showed cytotoxic and genotoxic effects in cultures, as well as necrosis, and increased inflammatory cells in the rabbit's tissues close to the implant.

Latrunculin A can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization.

PubMed

Terashita, Yukari; Wakayama, Sayaka; Yamagata, Kazuo; Li, Chong; Sato, Eimei; Wakayama, Teruhiko

2012-06-01

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (Citrus grandis) as a model. II. Cloning of resistance gene analogs from single chromosomes.

PubMed

Huang, D; Wu, W; Lu, L

2004-05-01

Amplification of resistance gene analogs (RGAs) is both a useful method for acquiring DNA markers closely linked to disease resistance (R) genes and a potential approach for the rapid cloning of R genes in plants. However, the screening of target sequences from among the numerous amplified RGAs can be very laborious. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed two methods for amplifying RGAs from single chromosomes. Method 1 uses products of Sau3A linker adaptor-mediated PCR (LAM-PCR) from a single chromosome as the templates for RGA amplification, while Method 2 directly uses a single chromosomal DNA molecule as the template. Using a pair of degenerate primers designed on the basis of the conserved nucleotide-binding-site motifs in many R genes, RGAs were successfully amplified from single chromosomes of pomelo using both these methods. Sequencing and cluster analysis of RGA clones obtained from single chromosomes revealed the number, type and organization of R-gene clusters on the chromosomes. We suggest that Method 1 is suitable for analyzing chromosomes that are unidentifiable under a microscope, while Method 2 is more appropriate when chromosomes can be clearly identified.

Clone-preventive technique that features magnetic microfibers and cryptography

NASA Astrophysics Data System (ADS)

Matsumoto, Hiroyuki; Suzuki, Keiichi; Matsumoto, Tsutomu

1998-04-01

We have used the term 'clone' to refer to those things which are produced by methods such as counterfeiting, alteration, duplication or simulation. To satisfy the requirements of secure and low-cost techniques for preventing card fraud, we have recently developed a clone preventive system called 'FibeCrypt (Fiber Cryptosystem)' which utilizes physical characteristics. Each card has a canonical domain (i.e. a distinctive part), similar to fingerprints as the biometric measurement, made up of magnetic micro-fibers scattered randomly inside. We have applied cryptosystems to the system. FibeCrypt examines and authenticates the unique pattern of the canonical domain using pre-stored reference data and a digital signature. In our paper, the schemes and the features of this system are described in detail. The results of our examinations show the accuracy of authentication of the system. We conclude that this authentication technique which utilizes physical characteristics can be very effective for clone prevention in various fields.

Computer network defense system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urias, Vincent; Stout, William M. S.; Loverro, Caleb

A method and apparatus for protecting virtual machines. A computer system creates a copy of a group of the virtual machines in an operating network in a deception network to form a group of cloned virtual machines in the deception network when the group of the virtual machines is accessed by an adversary. The computer system creates an emulation of components from the operating network in the deception network. The components are accessible by the group of the cloned virtual machines as if the group of the cloned virtual machines was in the operating network. The computer system moves networkmore » connections for the group of the virtual machines in the operating network used by the adversary from the group of the virtual machines in the operating network to the group of the cloned virtual machines, enabling protecting the group of the virtual machines from actions performed by the adversary.« less

Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

PubMed Central

Gole, Jeff; Gore, Athurva; Richards, Andrew; Chiu, Yu-Jui; Fung, Ho-Lim; Bushman, Diane; Chiang, Hsin-I; Chun, Jerold; Lo, Yu-Hwa; Zhang, Kun

2013-01-01

Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single E. coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1–2 Mb resolution. MIDAS will further the characterization of genomic diversity in many heterogeneous cell populations. PMID:24213699

Influence of regeneration method and tissue source on the frequency of somatic variation in Populus to infection by Septoria musiva

Treesearch

Michael E. Ostry; Ronald L. Hackett; Charles H. Michler; R. Serres; B. McCown

1994-01-01

Septoria leaf spot and canker are serious diseases of many hybrid poplar clones in plantations established for biomass production. Developing resistant clones through breeding is the best long-term strategy to minimize tree damage caused by this disease. Tissue culture and somaclonal selection techniques may reduce the time needed to develop disease resistance in...

Desensitization and Down Regulation of Muscarinic Acetylcholine Receptors

DTIC Science & Technology

1988-06-22

function, in vitro. This technique offers an easy method to obtain intact differentiated brain cells with minimal diffusion barriers. Preincubation of...neuroblastoma cells (clone NIE- 115 ). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one...mouse neuroblastoma NlE- 115 cells, and in other tissues, mediated an increase in phosphoinositide hydrolysis. Diacylglycerol is one of the important

Reproductive semi-cloning respecting biparental origin. A biologically unsound principle.

PubMed

Tateno, H; Latham, K E; Yanagimachi, R

2003-03-01

The original debate article proposed the use of "semi-cloning" as a viable method for assisted reproduction. This debate counters the proposal as being biologically unsound. Given the fundamental limitations of chromosomal segregation and genomic imprinting, the notion of using the MII oocyte to drive haploidization of a somatic cell genome and thereby obtain a substitute for authentic gametes is ill-conceived and untenable.

Population Diversity and Dynamics of Streptococcus mitis, Streptococcus oralis, and Streptococcus infantis in the Upper Respiratory Tracts of Adults, Determined by a Nonculture Strategy▿

PubMed Central

Bek-Thomsen, Malene; Tettelin, Hervé; Hance, Ioana; Nelson, Karen E.; Kilian, Mogens

2008-01-01

We reinvestigated the clonal diversity and dynamics of Streptococcus mitis and two other abundant members of the commensal microbiota of the upper respiratory tract, Streptococcus oralis and Streptococcus infantis, to obtain information about the origin of frequently emerging clones in this habitat. A culture-independent method was used, based on cloning and sequencing of PCR amplicons of the housekeeping gene gdh, which shows remarkable, yet species-specific, genetic polymorphism. Samples were collected from all potential ecological niches in the oral cavity and pharynx of two adults on two occasions separated by 2 years. Based on analysis of close to 10,000 sequences, significant diversity was observed in populations of all three species. Fluctuations in the relative proportions of individual clones and species were observed over time. While a few clones dominated, the proportions of most clones were very small. The results show that the frequent turnover of S. mitis, S. oralis, and S. infantis clones observed by cultivation can be explained by fluctuations in the relative proportions of clones, most of which are below the level of detection by the traditional culture technique, possibly combined with loss and acquisition from contacts. These findings provide a platform for understanding the mechanisms that govern the balance within the complex microbiota at mucosal sites and between the microbiota and the mucosal immune system of the host. PMID:18316382

SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea

PubMed Central

2010-01-01

Background Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process. Results Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots. Conclusions We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen. PMID:20359330

Two-Way Time Transfer via Geostationary Satellites NRC/NBS, NRC/USNO and NBS/USNO via Hermes and NRC/LPTF (France) via Symphonie

NASA Technical Reports Server (NTRS)

Costain, C.; Boulanger, J. S.; Daams, H.; Hanson, D. W.; Beehler, R. E.; Clements, A. J.; Davis, D. D.; Klepczynski, W. J.; Veenstra, L. B.; Kaiser, J.

1979-01-01

In most of the experiments, 1 pps pulses of the station atomic clocks were exchanged between the partners, and a cubic equation was fitted to the 1000 to 2000 second measurements. The equations were exchanged and substracted to obtain the time difference of the stations. The standard deviation in the fit of the equations varied, depending on conditions, from 1.5 ns to 16 ns. For the last month of the Hermes experiment, a 1 MHz signal was used, giving a standard deviation of 0.18 ns. The comparison of the time scales via satellite and via Loran-C (BIH Circular D) show clearly that some Loran-C links are very good, but that the NBS link varies by 1 micron s. Via the satellite the frequencies of the time scales can be compared with an accuracy of 2 x 10 to the minus 14 power.

Measurements of the cosmic background radiation

NASA Technical Reports Server (NTRS)

Lubin, P.; Villela, T.

1987-01-01

Maps of the large scale structure (theta is greater than 6 deg) of the cosmic background radiation covering 90 percent of the sky are now available. The data show a very strong 50-100 sigma (statistical error) dipole component, interpreted as being due to our motion, with a direction of alpha = 11.5 + or - 0.15 hours, sigma = -5.6 + or - 2.0 deg. The inferred direction of the velocity of our galaxy relative to the cosmic background radiation is alpha = 10.6 + or - 0.3 hours, sigma = -2.3 + or - 5 deg. This is 44 deg from the center of the Virgo cluster. After removing the dipole component, the data show a galactic signature but no apparent residual structure. An autocorrelation of the residual data, after substraction of the galactic component from a combined Berkeley (3 mm) and Princeton (12 mm) data sets, show no apparent structure from 10 to 180 deg with a rms of 0.01 mK(sup 2). At 90 percent confidence level limit of .00007 is placed on a quadrupole component.

Variable current speed controller for eddy current motors

DOEpatents

Gerth, H.L.; Bailey, J.M.; Casstevens, J.M.; Dixon, J.H.; Griffith, B.O.; Igou, R.E.

1982-03-12

A speed control system for eddy current motors is provided in which the current to the motor from a constant frequency power source is varied by comparing the actual motor speed signal with a setpoint speed signal to control the motor speed according to the selected setpoint speed. A three-phase variable voltage autotransformer is provided for controlling the voltage from a three-phase power supply. A corresponding plurality of current control resistors is provided in series with each phase of the autotransformer output connected to inputs of a three-phase motor. Each resistor is connected in parallel with a set of normally closed contacts of plurality of relays which are operated by control logic. A logic circuit compares the selected speed with the actual motor speed obtained from a digital tachometer monitoring the motor spindle speed and operated the relays to add or substract resistance equally in each phase of the motor input to vary the motor current to control the motor at the selected speed.

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

PubMed

Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

2013-01-30

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.

Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates

PubMed Central

Shimatsu, Yoshiki; Yamada, Kazuhiko; Horii, Wataru; Hirakata, Atsushi; Sakamoto, Yuji; Waki, Shiori; Sano, Junichi; Saitoh, Toshiki; Sahara, Hisashi; Shimizu, Akira; Yazawa, Hajime; Sachs, David H.; Nunoya, Tetsuo

2013-01-01

Background Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. Method and Results In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). Conclusions These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice. PMID:23581451

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

PubMed

Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

Fusarium diversity in soil using a specific molecular approach and a cultural approach.

PubMed

Edel-Hermann, Véronique; Gautheron, Nadine; Mounier, Arnaud; Steinberg, Christian

2015-04-01

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples. Published by Elsevier B.V.

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

PubMed

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.

PubMed

Günthard, H F; Wong, J K; Ignacio, C C; Havlir, D V; Richman, D D

1998-07-01

The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Temporal and intraclonal variation of flowering and pseudovivipary in Poa bulbosa

PubMed Central

Ofir, Micha; Kigel, Jaime

2014-01-01

Background and Aims Versatility in the reproductive development of pseudoviviparous grasses in response to growth conditions is an intriguing reproduction strategy. To better understand this strategy, this study examined variation in flowering and pseudovivipary among populations, co-occurring clones within populations, and among tillers in individual clones of Poa bulbosa, a summer-dormant geophytic grass that reproduces sexually by seed, and asexually by basal tiller bulbs and bulbils formed in proliferated panicles. Methods Clones were collected from 17 populations across a rainfall gradient. Patterns of reproduction were monitored for 11 years in a common garden experiment and related to interannual differences in climatic conditions. Intraclonal variation in flowering and pseudovivipary was studied in a phytotron, under daylengths marginal for flowering induction. Key Results Clones showed large temporal variability in their reproductive behaviour. They flowered in some years but not in others, produced normal or proliferated panicles in different years, or became dormant without flowering. Proliferating clones did not show a distinct time sequence of flowering and proliferation across years. Populations differed in incidence of flowering and proliferation. The proportion of flowering clones increased with decreasing rainfall at the site of population origin, but no consistent relationship was found between flowering and precipitation in the common garden experiment across years. In contrast, flowering decreased at higher temperatures during early growth stages after bulb sprouting. Pulses of soil fertilization greatly increased the proportion of flowering clones and panicle production. High intraclonal tiller heterogeneity was observed, as shown by the divergent developmental fates of daughter plants arising from bulbs from the same parent clone and grown under similar conditions. Panicle proliferation was enhanced by non-inductive 8 h short days, while marginally inductive 12 h days promoted normal panicles. Conclusions Interannual variation in flowering and proliferation in P. bulbosa clones was attributed to differences in the onset of the rainy season, resulting in different daylength and temperature conditions during the early stages of growth, during which induction of flowering and dormancy occurs. PMID:24685715

Can artificial parthenogenesis sidestep ethical pitfalls in human therapeutic cloning? An historical perspective

PubMed Central

Fangerau, H

2005-01-01

The aim of regenerative medicine is to reconstruct tissue that has been lost or pathologically altered. Therapeutic cloning seems to offer a method of achieving this aim; however, the ethical debate surrounding human therapeutic cloning is highly controversial. Artificial parthenogenesis—obtaining embryos from unfertilised eggs—seems to offer a way to sidestep these ethical pitfalls. Jacques Loeb (1859–1924), the founding father of artificial parthogenesis, faced negative public opinion when he published his research in 1899. His research, the public's response to his findings, and his ethical foundations serve as an historical argument both for the communication of science and compromise in biological research. PMID:16319240

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

PubMed Central

Lu, Wei; Liu, Jun; Xin, Qiang; Wan, Lili; Hong, Dengfeng; Yang, Guangsheng

2013-01-01

Background and Aims Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5. Methods A large F2 population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5. Key Results BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence. Conclusions This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus. PMID:23243189

Noise removal using factor analysis of dynamic structures: application to cardiac gated studies.

PubMed

Bruyant, P P; Sau, J; Mallet, J J

1999-10-01

Factor analysis of dynamic structures (FADS) facilitates the extraction of relevant data, usually with physiologic meaning, from a dynamic set of images. The result of this process is a set of factor images and curves plus some residual activity. The set of factor images and curves can be used to retrieve the original data with reduced noise using an inverse factor analysis process (iFADS). This improvement in image quality is expected because the inverse process does not use the residual activity, assumed to be made of noise. The goal of this work is to quantitate and assess the efficiency of this method on gated cardiac images. A computer simulation of a planar cardiac gated study was performed. The simulated images were added with noise and processed by the FADS-iFADS program. The signal-to-noise ratios (SNRs) were compared between original and processed data. Planar gated cardiac studies from 10 patients were tested. The data processed by FADS-iFADS were subtracted to the original data. The result of the substraction was studied to evaluate its noisy nature. The SNR is about five times greater after the FADS-iFADS process. The difference between original and processed data is noise only, i.e., processed data equals original data minus some white noise. The FADS-iFADS process is successful in the removal of an important part of the noise and therefore is a tool to improve the image quality of cardiac images. This tool does not decrease the spatial resolution (compared with smoothing filters) and does not lose details (compared with frequential filters). Once the number of factors is chosen, this method is not operator dependent.

Cloning Mice.

PubMed

Ogura, Atsuo

2017-08-01

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos. © 2017 Cold Spring Harbor Laboratory Press.

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system

PubMed Central

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

2006-01-01

OBJECTIVE To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. METHODS Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. RESULTS Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. CONCLUSION The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease. PMID:18651010

Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal

PubMed Central

Hamad, Ibrahim; Sokhna, Cheikh; Raoult, Didier; Bittar, Fadi

2012-01-01

Background Microbial eukaryotes represent an important component of the human gut microbiome, with different beneficial or harmful roles; some species are commensal or mutualistic, whereas others are opportunistic or parasitic. The diversity of eukaryotes inhabiting humans remains relatively unexplored because of either the low abundance of these organisms in human gut or because they have received limited attention from a whole-community perspective. Methodology/Principal Finding In this study, a single fecal sample from a healthy African male was studied using both culture-dependent methods and extended molecular methods targeting the 18S rRNA and ITS sequences. Our results revealed that very few fungi, including Candida spp., Galactomyces spp., and Trichosporon asahii, could be isolated using culture-based methods. In contrast, a relatively a high number of eukaryotic species could be identified in this fecal sample when culture-independent methods based on various primer sets were used. A total of 27 species from one sample were found among the 977 analyzed clones. The clone libraries were dominated by fungi (716 clones/977, 73.3%), corresponding to 16 different species. In addition, 187 sequences out of 977 (19.2%) corresponded to 9 different species of plants; 59 sequences (6%) belonged to other micro-eukaryotes in the gut, including Entamoeba hartmanni and Blastocystis sp; and only 15 clones/977 (1.5%) were related to human 18S rRNA sequences. Conclusion Our results revealed a complex eukaryotic community in the volunteer’s gut, with fungi being the most abundant species in the stool sample. Larger investigations are needed to assess the generality of these results and to understand their roles in human health and disease. PMID:22808282

Revival of extinct species using nuclear transfer: hope for the mammoth, true for the Pyrenean ibex, but is it time for "conservation cloning"?

PubMed

Piña-Aguilar, Raul E; Lopez-Saucedo, Janet; Sheffield, Richard; Ruiz-Galaz, Lilia I; Barroso-Padilla, Jose de J; Gutiérrez-Gutiérrez, Antonio

2009-09-01

Recent accomplishments in the fields of nuclear transfer and genomics, such as the cloned offspring production from frozen mouse cells, cryopreserved at not too low temperatures without cryoprotectors; or the sequencing of wooly mammoth genome, have opened the opportunity for the revival of extinct species. As expected, they are receiving a lot of publicity in the media and also scientific attention. Furthermore, it was recently published the "revival" of the first extinct subspecie: the Pyrenean ibex (Capra pyrenaica pyrenaica), a wild goat extinct in 2000. This strengthens the field of cloning as it had been tarnished by induced pluripotent stem cells (iPS) and other methods of reprogramming. However, for biological conservation purposes, cloning is not generally accepted as an alternative for animal conservation, and there is an ongoing debate between reproductive scientists and conservation specialists. Although we believe that nuclear transfer technologies have an opportunity in conservation efforts for some species that are on the brink of extinction and that population status, geographical isolation, reproductive characteristics, and human pressure create a situation that is almost unsustainable. In this article we discuss the barriers in cloning mammoths and cloning controversies in conservation from a zoological perspective, citing the species that might benefit from nuclear transfer techniques in the arduous journey so as not to disappear forever from this, our world.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique.

PubMed

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A; Gonzalez, Luis; Martin, Luis M; Miranda, Jorge; Navarro-Cerrillo, Rafael M

2009-02-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique*

PubMed Central

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A.; Gonzalez, Luis; Martin, Luis M.; Miranda, Jorge; Navarro-Cerrillo, Rafael M.

2009-01-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin. PMID:19235269

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

PubMed Central

Hietala, Ari M.; Eikenes, Morten; Kvaalen, Harald; Solheim, Halvor; Fossdal, Carl G.

2003-01-01

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance. PMID:12902224

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1)

PubMed Central

Ahmad, Muhammad Khairi; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Sandai, Doblin Anak; Mohamed, Rafeezul; Ismail, Ida Shazrina; Zulkiflie, Nurulisa; Yunus, Muhammad Amir

2017-01-01

Background A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. Methods The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3′end of the reporter gene and the VP2 start sequence to allow co-translational ‘cleavage’ of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Results Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. Conclusion NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication. PMID:29379384

High-throughput gene mapping in Caenorhabditis elegans.

PubMed

Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

2002-07-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

PubMed

Wang, Fen; Ye, Bin

2016-10-01

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

PubMed

Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

PubMed Central

Hudson, Elton P.; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications. PMID:22624028

Hypoxia enhances periodontal ligament stem cell proliferation via the MAPK signaling pathway.

PubMed

He, Y; Jian, C X; Zhang, H Y; Zhou, Y; Wu, X; Zhang, G; Tan, Y H

2016-11-21

There is high incidence of periodontal disease in high-altitude environments; hypoxia may influence the proliferation and clone-forming ability of periodontal ligament stem cells (PDLSCs). The MAPK signaling pathway is closely correlated with cell proliferation, differentiation, and apoptosis. Thus, we isolated and cultured PDLSCs under hypoxic conditions to clarify the impact of hypoxia on PDLSC proliferation and the underlying mechanism. PDLSCs were separated and purified by the limiting dilution method and identified by flow cytometry. PDLSCs were cultured under hypoxic or normoxic conditions to observe their cloning efficiency. PDLSC proliferation at different oxygen concentrations was evaluated by MTT assay. Expression of p38/MAPK and MAPK/ERK signaling pathway members was detected by western blotting. Inhibitors for p38/MAPK or ERK were applied to PDLSCs to observe their impacts on clone formation and proliferation. Isolated PDLSCs exhibited typical stem cell morphological characteristics, strong abilities of globular clone formation and proliferation, and upregulated expression of mesenchymal stem cell markers. Stem cell marker expression was not statistically different between PDLSCs cultured under hypoxia and normoxia (P > 0.05). The clone number in the hypoxia group was significantly higher than that in the control (P < 0.05). PDLSC proliferation under hypoxia was higher than that of the control (P < 0.001). p38 and ERK1/2 phosphorylation in hypoxic PDLSCs was markedly enhanced compared to that in the control (P < 0.05). Either P38/MAPK inhibitor or ERK inhibitor treatment reduced clone formation and proliferation. Therefore, hypoxia enhanced PDLSC clone formation and proliferation by activating the p38/MAPK and ERK/MAPK signaling pathways.

Taxonomic and functional assignment of cloned sequences from high Andean forest soil metagenome.

PubMed

Montaña, José Salvador; Jiménez, Diego Javier; Hernández, Mónica; Angel, Tatiana; Baena, Sandra

2012-02-01

Total metagenomic DNA was isolated from high Andean forest soil and subjected to taxonomical and functional composition analyses by means of clone library generation and sequencing. The obtained yield of 1.7 μg of DNA/g of soil was used to construct a metagenomic library of approximately 20,000 clones (in the plasmid p-Bluescript II SK+) with an average insert size of 4 Kb, covering 80 Mb of the total metagenomic DNA. Metagenomic sequences near the plasmid cloning site were sequenced and them trimmed and assembled, obtaining 299 reads and 31 contigs (0.3 Mb). Taxonomic assignment of total sequences was performed by BLASTX, resulting in 68.8, 44.8 and 24.5% classification into taxonomic groups using the metagenomic RAST server v2.0, WebCARMA v1.0 online system and MetaGenome Analyzer v3.8 software, respectively. Most clone sequences were classified as Bacteria belonging to phlya Actinobacteria, Proteobacteria and Acidobacteria. Among the most represented orders were Actinomycetales (34% average), Rhizobiales, Burkholderiales and Myxococcales and with a greater number of sequences in the genus Mycobacterium (7% average), Frankia, Streptomyces and Bradyrhizobium. The vast majority of sequences were associated with the metabolism of carbohydrates, proteins, lipids and catalytic functions, such as phosphatases, glycosyltransferases, dehydrogenases, methyltransferases, dehydratases and epoxide hydrolases. In this study we compared different methods of taxonomic and functional assignment of metagenomic clone sequences to evaluate microbial diversity in an unexplored soil ecosystem, searching for putative enzymes of biotechnological interest and generating important information for further functional screening of clone libraries.

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Chiara, Gabriele; Fazio, Rosario; Montangero, Simone

In this paper we present an approach to quantum cloning with unmodulated spin networks. The cloner is realized by a proper design of the network and a choice of the coupling between the qubits. We show that in the case of phase covariant cloner the XY coupling gives the best results. In the 1{yields}2 cloning we find that the value for the fidelity of the optimal cloner is achieved, and values comparable to the optimal ones in the general N{yields}M case can be attained. If a suitable set of network symmetries are satisfied, the output fidelity of the clones doesmore » not depend on the specific choice of the graph. We show that spin network cloning is robust against the presence of static imperfections. Moreover, in the presence of noise, it outperforms the conventional approach. In this case the fidelity exceeds the corresponding value obtained by quantum gates even for a very small amount of noise. Furthermore, we show how to use this method to clone qutrits and qudits. By means of the Heisenberg coupling it is also possible to implement the universal cloner although in this case the fidelity is 10% off that of the optimal cloner.« less

Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

NASA Astrophysics Data System (ADS)

Chen, Ellson Y.

1997-05-01

So far we have used OSS strategy to sequence over 2 megabases DNA in large-insert clones from regions of human X chromosomes with different characteristic levels of GC content. The method starts by randomly fragmenting a BAC, YAC or PAC to 8-12 kb pieces and subcloning those into lambda phage. Insert-ends of these clones are sequenced and overlapped to create a partial map. Complete sequencing is then done on a minimal tiling path of selected subclones, recursively focusing on those at the edges of contigs to facilitate mergers of clones across the entire target. To reduce manual labor, PCR processes have been adapted to prepare sequencing templates throughout the entire operation. The streamlined process can thus lend itself to further automation. The OSS approach is suitable for large- scale genomic sequencing, providing considerable flexibility in the choice of subclones or regions for more or less intensive sequencing. For example, subclones containing contaminating host cell DNA or cloning vector can be recognized and ignored with minimal sequencing effort; regions overlapping a neighboring clone already sequenced need not be redone; and segments containing tandem repeats or long repetitive sequences can be spotted early on and targeted for additional attention.

Use of the piggyBac transposon to create stable packaging cell lines for the production of clinical-grade self-inactivating γ-retroviral vectors.

PubMed

Feldman, Steven A; Xu, Hui; Black, Mary A; Park, Tristen S; Robbins, Paul F; Kochenderfer, James N; Morgan, Richard A; Rosenberg, Steven A

2014-08-01

Efforts to improve the biosafety of γ-retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-retroviral vectors. However, scale-up and manufacturing of such vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-retroviral vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-retroviral vectors for ex vivo gene therapy clinical trials.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

PubMed Central

Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu

2017-01-01

ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method.

PubMed

Lee, Seunghoon; Zhao, Minghui; No, Jingu; Nam, Yoonseok; Im, Gi-Sun; Hur, Tai-Young

2017-01-01

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6-15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method

PubMed Central

No, Jingu; Nam, Yoonseok; Im, Gi-Sun; Hur, Tai-Young

2017-01-01

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning. PMID:28288197

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2014-06-01

Specifically, we combined the CRISPR genome editing system with a novel approach allowing efficient single cell cloning of Drosophila cells with the aim of...and culture these to produce cultures completely lacking wildtype sequence at the target locus. No robust methods existed to clone single Drosophila ...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . 65 samples that displayed synthetic lethality (15 genes) or synthetic

What is Cloning?

MedlinePlus

Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

[Clonal variability in longevity of the cercariae of Himasthla elongata (Trematoda: Echinostomatidae)].

PubMed

Levakin, I A; Losev, E A; Zavirskiĭ, Ia V; Galaktionov, K V

2013-01-01

The study was carried out on Himasthla elongata cercariae shed by infected Littorina littorea snails. The infected periwinkles were collected from the settlement with the low prevalence of H. elongata. As shown earlier with the use of AFLP (Amplified Fragment Length Polymorphisms) method, rediae groups in all the infected periwinkles of this settlement arise from the infection of a mollusc with a single miracidium. Therefore, the cercariae shed by an infected mollusc have the same genotype or, in other words, represent a clone. The LT50 (the time during which 50% of cercariae perish in the experimental dish) were measured experimentally for cercariae Himasthla elongata belong to different clones. The investigated parameter demonstrated a high level of interclonal variability. Two groups of cercarial clones were identified: one of them was characterized by the high level of intraclonal variability in LT50 and the second, by the low one. It is assumed that the observed heterogeneity may be stipulated by different degrees of mitotic recombinations during formation of different cercarial clones.

Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

PubMed Central

Lin, Jinke; Kudrna, Dave; Wing, Rod A.

2011-01-01

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers. PMID:21234344

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sciarrino, Fabio; Dipartimento di Fisica and Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, Universita 'La Sapienza', Rome 00185; De Martini, Francesco

The optimal phase-covariant quantum cloning machine (PQCM) broadcasts the information associated to an input qubit into a multiqubit system, exploiting a partial a priori knowledge of the input state. This additional a priori information leads to a higher fidelity than for the universal cloning. The present article first analyzes different innovative schemes to implement the 1{yields}3 PQCM. The method is then generalized to any 1{yields}M machine for an odd value of M by a theoretical approach based on the general angular momentum formalism. Finally different experimental schemes based either on linear or nonlinear methods and valid for single photon polarizationmore » encoded qubits are discussed.« less

Cloning of polymorphisms (COP): enrichment of polymorphic sequences from complex genomes

PubMed Central

Li, Jingfeng; Wang, Fuli; Zabarovska, Veronika; Wahlestedt, Claes; Zabarovsky, Eugene R.

2000-01-01

Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed. PMID:10606669

Clinical impact of methicillin-resistant staphylococcus aureus on bacterial pneumonia: cultivation and 16S ribosomal RNA gene analysis of bronchoalveolar lavage fluid.

PubMed

Kawanami, Toshinori; Yatera, Kazuhiro; Yamasaki, Kei; Noguchi, Shingo; Fukuda, Kazumasa; Akata, Kentarou; Naito, Keisuke; Kido, Takashi; Ishimoto, Hiroshi; Taniguchi, Hatsumi; Mukae, Hiroshi

2016-04-16

Determining whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen or reflective of colonization when MRSA is cultured from the respiratory tract remains important in treating patients with pneumonia. We evaluated the bacterial microbiota in bronchoalveolar lavage fluid (BALF) using the clone library method with a 16S ribosomal RNA (rRNA) gene analysis in 42 patients from a pneumonia registry who had MRSA cultured from their sputum or BALF samples. Patients were divided into two groups: those treated with (Group A) or without (Group B) anti-MRSA agents, and their clinical features were compared. Among 248 patients with pneumonia, 42 patients who had MRSA cultured from the respiratory tract were analyzed (Group A: 13 patients, Group B: 29 patients). No clones of S. aureus were detected in the BALF of 20 out of 42 patients. Twenty-eight of 29 patients in Group B showed favorable clinical outcomes, indicating that these patients had non-MRSA pneumonia. Using a microflora analysis of the BALF, the S. aureus phylotype was predominant in 5 of 28 (17.9%) patients among the detected bacterial phylotypes, but a minor population (the percentage of clones ≤ 10%) in 19 (67.9%) of 28 patients. A statistical analysis revealed no positive relationship between the percentage of clones of the S. aureus phylotype and risk factors of MRSA pneumonia. The molecular method using BALF specimens suggests that conventional cultivation method results may mislead true causative pathogens, especially in patients with MRSA pneumonia. Further studies are necessary to elucidate these clinically important issues.

Molecular characterization and antibiotic susceptibility of Haemophilus influenzae clinical isolates.

PubMed

K L Ç, Hüseyin; Akyol, Selcan; Parkan, Õmür Mustafa; Dinç, Gõkçen; Sav, Hafize; Aydemir, Gonca

2017-03-01

Haemophilus influenzae can cause invasive and severe infections in both adults and children such as otitis media, sinusitis, pneumonia, meningitis and bacteremia. The emerging antibiotic resistance in recent years against ampicillin and several other antibiotics among strains of H. influenzae gives cause for serious concern. Here, we investigate ß-lactamase (BL) activity in clinical isolates of H. influenzae, profile their resistance to antibiotics, and characterize the clonal relationship of the isolates. Antibiotic susceptibilities of 92 clinical isolates of H. influenzae (March 2011-May 2012) were determined using the disk diffusion method according to the Clinical & Laboratory Standards Institute (CLSI), and BL activity was detected using the nitrocefin disk method. The Rep-PCR method was used to characterize clonality of the isolates. All strains were found to be susceptible to levofloxacin and cefotaxime. Four isolates out of 92 (4.3%) were found resistant to ampicillin, one isolate (1.1%) was resistant to amoxicillin/clavulanic acid, 21 isolates (22.8%) were resistant to trimethoprim-sulfamethoxazole (SXT), and three isolates (3.3%) showed BL activity. One strain was BL-negative but resistant to ampicillin. The three isolates with BL activity and four isolates with resistance to ampicillin did not have a clonal relationship. Three distinct clones [clone A (with subclones A1 and A2), clone B, and clone C] were identified among the SXT-resistant strains. Most of the H. influenzae isolates in this study were susceptible to the antibiotics while SXT resistance was relatively more prevalent, which suggests that significant obstacles in the therapeutic use of antibiotics against H. influenzae strains are not expected in our region.

Methods of preparing and using single chain anti-tumor antibodies

DOEpatents

Cheung, Nai-Kong; Guo, Hong-Fen

2010-02-23

This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

Method for preparation of single chain antibodies

DOEpatents

Cheung, Nai-Kong V [New York, NY; Guo, Hong-fen [New York, NY

2012-04-03

This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

PubMed

Maruthamuthu, Mukil; Jiménez, Diego Javier; Stevens, Patricia; van Elsas, Jan Dirk

2016-01-28

Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications.

Chromatographic analysis of salicylic compounds in different species of the genus Salix.

PubMed

Pobłocka-Olech, Loretta; van Nederkassel, Anne-Marie; Vander Heyden, Yvan; Krauze-Baranowska, Mirosława; Glód, Daniel; Baczek, Tomasz

2007-11-01

The separation of nine phenol glycosides--salicin, salicortin, 2'-acetylsalicortin, populin, tremulacin, salidroside, triandrin, picein and helicin--by normal phase (NP), reversed phase (RP) HPLC techniques and a coupling of NP and RP monolithic silica columns was studied. Among the above nine compounds only five--salicin, populin, tremulacin, salidroside and triandrin--were resolved in an NP system with a mobile phase comprising hexane/isopropanol/methanol (87:12:1, v/v/v). Optimized separation was performed with two coupled monolithic silica columns of different polarity (bare silica and RP-18). The method was applied to verify the presence of salicylic compounds and other phenolic derivatives in the bark of six species from the genus Salix, namely S. purpurea, S. daphnoides clone 1095, S. alba clone 1100, S. triandra, S. viminalis, and S. herbacea. Gradient elution with a mobile phase composed of acetonitrile and water containing 0.05% of trifluoroacetic acid, with increasing acetonitrile concentration from 3% to 48%, was chosen as optimal. For the selective detection of the salicylic compounds, an evaporative light scattering detector was employed along with a UV detector. The differences in the composition of phenols in the different plant materials were confirmed. Additionally, it must be emphasized that for the first time the presence of 2'-acetylsalicortin was revealed in S. alba clone 1100. Furthermore, an SPE-HPLC method was developed for the rapid analysis of the salicin content, analyzed as free and total fraction, in willow barks. The determined concentrations of total salicin varied from 25.4 mg/g in S. alba clone 1100 to 96.47 mg/g in S. daphnoides clone 1095.

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1991-01-21

bisphosphate carboxylase ( RubisCO ) from symbiotic bacteria of various origins, b) To continue methods development for 16S rRNA sequencing from symbionts in...frozen and badly preserved specimens, and c) To use these new techniques to sequence 16s DNA from a variety of symbionts a) RubisCO We have cloned the...gene coding for RubisCO from the sulfur oxidixing symbiont of the gastropod Alvinochoncha hessleri. Nucleotide sequence analysis of the cloned fragment

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

PubMed

Rangnoi, Kuntalee; Choowongkomon, Kiattawee; O'Kennedy, Richard; Rüker, Florian; Yamabhai, Montarop

2018-06-06

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

PubMed

Shukla, Girja S; Krag, David N; Peletskaya, Elena N; Pero, Stephanie C; Sun, Yu-Jing; Carman, Chelsea L; McCahill, Laurence E; Roland, Thomas A

2013-08-01

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

PubMed Central

Liu, X; Gorovsky, M A

1996-01-01

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

Physiological and biochemical responses to severe drought stress of nine Eucalyptus globulus clones: a multivariate approach.

PubMed

Granda, Víctor; Delatorre, Carolina; Cuesta, Candela; Centeno, María L; Fernández, Belén; Rodríguez, Ana; Feito, Isabel

2014-07-01

Seasonal drought, typical of temperate and Mediterranean environments, creates problems in establishing plantations and affects development and yield, and it has been widely studied in numerous species. Forestry fast-growing species such as Eucalyptus spp. are an important resource in such environments, selected clones being generally used for production purposes in plantations in these areas. However, use of mono-specific plantations increases risk of plant loss due to abiotic stresses, making it essential to understand differences in an individual clone's physiological responses to drought stress. In order to study clonal differences in drought responses, nine Eucalyptus globulus (Labill.) clones (C14, C46, C97, C120, C222, C371, C405, C491 and C601) were gradually subjected to severe drought stress (<14% of field capacity). A total of 31 parameters, physiological (e.g., photosynthesis, gas exchange), biochemical (e.g., chlorophyll content) and hormonal (abscisic acid [ABA] content), were analysed by classic and multivariate techniques. Relationships between parameters were established, allowing related measurements to be grouped into functional units (pigment, growth, water and ABA). Differences in these units showed that there were two distinct groups of E. globulus clones on the basis of their different strategies when faced with drought stress. The C14 group (C14, C120, C405, C491 and C601) clones behave as water savers, maintaining high water content and showing high stomatal adjustment, and reducing their aerial growth to a great extent. The C46 group (C46, C97, C222 and C371) clones behave as water spenders, reducing their water content drastically and presenting osmotic adjustment. The latter maintains the highest growth rate under the conditions tested. The method presented here can be used to identify appropriate E. globulus clones for drought environments, facilitating the selection of material for production and repopulation environments. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A new method for predicting crashworthiness.

PubMed

Council, F M; Stewart, J R; Cox, C L

1997-01-01

Consumer information concerning the predicted 'safeness' of a new car model is based on the results of crash tests. Unfortunately, because it allows comparisons only within size/weight groups, the information is somewhat incompatible with the normal car-purchase decision process since consumers often consider cars within different groups. In addition, based on past research, the association of the crash-test information with real-world crash outcomes is, at best, somewhat limited. The goal of this study was to explore a methodology for improving this information, a methodology which incorporates not only the crash-test information, but also information concerning real-world occupant injury experience in prior crashes involving similar vehicles ('clones'). The clone information included both driver injury severity in past clone crashes from the North Carolina accident file and various indicators of relative driver injury in clones extracted from published insurance-related data from the Highway Loss Data Institute (HLDI). Final models developed included both measures of the Head Index Criteria (HIC) from the crash test and some measure of clone performances as significant predictors. While the North Carolina clone data is intuitively 'cleaner' in that it describes injury level per crash rather than per insured year, the medical claims indices from the HLDI data consistently were shown to be the stronger predictors. Future research will need to look at ways of better combining the crash-test variables and of possible modifications to the HLDI indices. In general, the analyses generated encouraging results that appear to point to possible improvements in the crashworthiness information.

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

PubMed Central

Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

1997-01-01

mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology.

PubMed

Yu, Deshui; Liao, Libing; Zhang, Ju; Zhang, Yi; Xu, Kedong; Liu, Kun; Li, Xiaoli; Tan, Guangxuan; Chen, Ran; Wang, Yulu; Liu, Xia; Zhang, Xuan; Han, Xiaomeng; Wei, Zhangkun; Li, Chengwei

2018-05-01

Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

[Growth analysis on modules of Cynodon dactylon clones in Yili River Valley Plain of Xinjiang].

PubMed

Zhao, Yu; Janar; Li, Hai-Yan; Liu, Ying; Yang, Yun-Fei

2009-04-01

By the method of randomly digging up whole ramet tuft while maintaining natural integrity, large samples of Cynodon dactylon clones were collected from a grape orchard abandoned for 2 years without any management in the Yili River Valley Plain of Xinjiang, aimed to quantitatively analyze the growth patterns of their modules. The results showed that the average ramet number of test 30 clones reached 272.6 +/- 186. 6, among which, vegetative ramets occupied 82.3%, being 4.3 times higher than reproductive ones. The total biomass of the clones was 45.4 +/- 40.0 g, in which, rhizomes accounted for 54.4%, while the vegetative ramets, stolons, and reproductive ramets occupied 21.0%, 14.8%, and 9.4% of the total, respectively. The accumulative length of rhizomes and stolons reached 5.1 + 4.7 m and 3.3 +/- 3.4 m, while the bud number on stolons and rhizomes was 291.5 +/- 246.8 and 78.8 +/- 87.4, respectively. The bud number on stolons and rhizomes was positively correlated to the quantitative characters of vegetative ramets, reproductive ramets, stolons, and rhizomes (P < 0.01), indicating that in Yili River Valley Plain, C. dactylon clone could achieve and maintain its continuous renovation via rhizome buds.

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy

PubMed Central

Levin, Mattias; King, Jasmine J.; Glanville, Jacob; Jackson, Katherine J. L.; Looney, Timothy J.; Hoh, Ramona A.; Mari, Adriano; Andersson, Morgan; Greiff, Lennart; Fire, Andrew Z.; Boyd, Scott D.; Ohlin, Mats

2016-01-01

Background Specific immunotherapy (SIT) is the only treatment with proven long-term curative potential in allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B cell repertoires are not well understood. Objective To characterize the IgE sequences expressed by allergen-specific B cells, and track the fate of these B cell clones during SIT. Methods We have used high-throughput antibody gene sequencing and identification of allergen-specific IgE using combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and nasal mucosa of aeroallergen-sensitized individuals before and during the first year of subcutaneous SIT. Results Of 52 distinct allergen-specific IgE heavy chains from eight allergic donors, 37 were also detected by high-throughput antibody gene sequencing of blood, nasal mucosa, or both sample types. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. Conclusion Combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies may in the future aid assessment of SIT mechanisms and efficacy. PMID:26559321

Aggregates, Crystals, Gels, and Amyloids: Intracellular and Extracellular Phenotypes at the Crossroads of Immunoglobulin Physicochemical Property and Cell Physiology

PubMed Central

2013-01-01

Recombinant immunoglobulins comprise an important class of human therapeutics. Although specific immunoglobulins can be purposefully raised against desired antigen targets by various methods, identifying an immunoglobulin clone that simultaneously possesses potent therapeutic activities and desirable manufacturing-related attributes often turns out to be challenging. The variable domains of individual immunoglobulins primarily define the unique antigen specificities and binding affinities inherent to each clone. The primary sequence of the variable domains also specifies the unique physicochemical properties that modulate various aspects of individual immunoglobulin life cycle, starting from the biosynthetic steps in the endoplasmic reticulum, secretory pathway trafficking, secretion, and the fate in the extracellular space and in the endosome-lysosome system. Because of the diverse repertoire of immunoglobulin physicochemical properties, some immunoglobulin clones' intrinsic properties may manifest as intriguing cellular phenotypes, unusual solution behaviors, and serious pathologic outcomes that are of scientific and clinical importance. To gain renewed insights into identifying manufacturable therapeutic antibodies, this paper catalogs important intracellular and extracellular phenotypes induced by various subsets of immunoglobulin clones occupying different niches of diverse physicochemical repertoire space. Both intrinsic and extrinsic factors that make certain immunoglobulin clones desirable or undesirable for large-scale manufacturing and therapeutic use are summarized. PMID:23533417

CD34 expression in human hair follicles and tricholemmoma: a comprehensive study.

PubMed

Misago, Noriyuki; Toda, Shuji; Narisawa, Yutaka

2011-08-01

There has recently been controversy regarding whether clone My10 is superior to clone QBEND-10 for labeling cells of tricholemmal lineage. Moreover, there have been no previous reports on the CD34 expression in human vellus hair follicles. We performed a comprehensive study of the CD34 expression in human terminal and vellus hair follicles and in 10 tricholemmomas using both the QBEND-10 and the My10 clones. We also performed two different procedures of immunostaining, which included the using of the standard avidin-biotin-peroxidase (ABC) complex system and the Envision system. The most sensitive marker of CD34 for normal human hair follicles and tricholemmomas is QBEND-10 using the ABC system. The degree and strength of the CD34 positive staining mainly depended on the method being used (whether it was the ABC system or the Envision system) rather than the clone. CD34 staining was rarely (20-30%) seen in the anagen and catagen vellus hair follicles, and could only be seen by the QBEND-10 clone using the ABC system. CD34 expression in the tricholemmomas represented either a diffuse or peripheral pattern. CD34 may not be a tricholemmal lineage-specific antigen, but may be related to certain functions of the cells. Copyright © 2011 John Wiley & Sons A/S.

A cloned DNA segment from the telomeric region of human chromosome 4p is not detectably rearranged in Huntington disease patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pritchard, C.; Casher, D.; Myers, R.M.

1990-09-01

Genetic linkage studies have mapped the Huntington disease (HD) mutation to the distal region of the short arm of human chromosome 4. Analysis of recombination events in this region has produced contradictory locations for HD. One possible location is in the region distal to the D4S90 marker, which is located within 300 kilobases of the telomere. Other crossover events predict a more centromeric position for HD. Here the authors analyze the telomeric region of 4p in detail. Cloned DNA segments were derived from this region by utilizing a radiation-induced somatic cell hybrid as a source of DNA combined with preparativemore » pulsed-field gel electrophoresis to enrich for the telmoeric fraction. Additional DNA was obtained by using the cloned segments as multiple start points for cosmid walks. This strategy proved to be an effective method for cloning 250 kilobases of DNA in the region telomeric to D4S90. Hybridization analysis with the cloned DNA did not provide any evidence for the presence of rearrangements of 100 base pairs or greater in the DNA of individuals affected with HD. They also found no charge in the size or structure of the 4p telomere in these samples.« less

Ethical, legal, and social aspects of farm animal cloning in the 6th Framework Programme for Research.

PubMed

Claxton, John; Sachez, Elena; Matthiessen-Guyader, Line

2004-01-01

Cloned livestock have potential importance in the provision of improved medicine as well as in the development of livestock production. The public is, however, increasingly concerned about the social and ethical consequences of these advances in knowledge and techniques. There is unevenness throughout Europe in different Member States' attitudes to research into livestock cloning. Although there is EU legislation controlling the use of animals for research purposes, there is no legislation specifically governing cloning in livestock production. The main EU reference is the 9th Opinion of the European Group on Ethics, which states "Cloning of farm animals may prove to be of medical and agricultural as well as economic benefit. It is acceptable only when the aims and methods are ethically justified and when carried out under ethical conditions." The ethical justification includes the avoidance of suffering, the use of the 3Rs principle and a lack of better alternatives. The Commission addresses these issues in the 6th Framework Programme by promoting the integration of ethical, legal and social aspects in all proposals where they are relevant, by fostering ethical awareness and foresight in the proposals, by encouraging public dialogue, and by supporting specific actions to promote the debate. Research must respect fundamental ethical principles, including animal welfare requirements.

Cloning of ES cells and mice by nuclear transfer.

PubMed

Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2009-01-01

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers.

PubMed

Howland, Shanshan W; Poh, Chek-Meng; Rénia, Laurent

2011-09-01

Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5'→3' exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

Biochemical fingerprinting of Vibrio parahaemolyticus by the PhenePlate system: comparison between pandemic and non-pandemic serotypes.

PubMed

Rahman, Mokhlasur; Bhuiyan, N A; Kuhn, I; Ramamurthy, T; Rahman, M; Mollby, R; Nair, G Balakrish

2006-10-01

During recent years a pandemic clone of Vibrio parahaemolyticus has emerged. Isolates of this clone are distributed among several serotypes, but are genotypically related. In the present study, a phenotyping method (biochemical fingerprinting) was used to characterize pandemic and non-pandemic isolates belonging to V. parahaemolyticus. It was found that the pandemic isolates showed a high level of phenotypic homogeneity and a majority of the pandemic isolates belonged to the same biochemical phenotype, whereas non-pandemic V. parahemolyticus isolates were more heterogeneous. In conclusion, biochemical fingerprinting of V. parahaemolyticus can be used as a first screening method to differentiate between pandemic and non-pandemic isolates of V. parahaemolyticus.

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

PubMed Central

Zampini, Massimiliano; Stevens, Pauline Rees; Pachebat, Justin A.; Kingston-Smith, Alison; Mur, Luis A. J.; Hayes, Finbarr

2015-01-01

The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli. PMID:26062748

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, J.J.; Quesada, M.A.; Randesi, M.

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector. The cloning vector has an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Method for producing labeled single-stranded nucleic acid probes

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-10-19

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Three concepts of cloning in human beings.

PubMed

Cui, Ke-Hui

2005-07-01

Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

DTIC Science & Technology

1988-02-02

the bands excised, and the DNA extracted with phenol for cloning in M13 . 6 Nuclotida sequence analysis. The two fragments were each cloned into phages ...DNA; and strain JM103 (29) was used to propagate M13 ph&ge derivatives. -1 Subcloning and detection of PA-producing rsccmbinants. The isolation of...method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 19. Lauben, J. 0., and J. 2. K. Nielsen. 1982. Penicillinase and

Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

PubMed

Nomoto, Mika; Tada, Yasuomi

2018-01-01

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

PubMed

Charles, Edgar D; Orloff, Michael I M; Dustin, Lynn B

2011-01-05

Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 μg/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance. Copyright © 2010 Elsevier B.V. All rights reserved.

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing.

PubMed

Giraud, Mathieu; Salson, Mikaël; Duez, Marc; Villenet, Céline; Quief, Sabine; Caillault, Aurélie; Grardel, Nathalie; Roumier, Christophe; Preudhomme, Claude; Figeac, Martin

2014-05-28

V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols. The proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast.

PubMed

Seo, Hogyu David; Lee, Daeyoup

2018-05-15

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

PubMed Central

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

Microorganism genomics, compositions and methods related thereto

DOEpatents

Handelsman, Jo; Goodman, Robert M.; Rondon, Michelle R.

2001-01-01

The present invention provides methods and compositions for accessing, in a generally unbaised manner, a diverse genetic pool for genes involved in biosynthetic pathways. The invention also provides compounds which can be identified by cloning biosynthetic pathways.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

PubMed

Butterworth, Alice S; Robertson, Alan J; Ho, Mei-Fong; Gatton, Michelle L; McCarthy, James S; Trenholme, Katharine R

2011-04-18

Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue. © 2011 Butterworth et al; licensee BioMed Central Ltd.

Persons and their copies.

PubMed Central

McCarthy, D

1999-01-01

Is cloning human beings morally wrong? The basis for the one serious objection to cloning is that, because of what a clone is, clones would have much worse lives than non-clones. I sketch a fragment of moral theory to make sense of the objection. I then outline several ways in which it might be claimed that, because of what a clone is, clones would have much worse lives than non-clones. In particular, I look at various ideas connected with autonomy. I conclude that there is no basis to the claim that, because of what a clone is, clones would have much worse lives than non-clones. I therefore reject the claim that cloning human beings is morally wrong. PMID:10226912

Transmitter responsiveness in two newly isolated clones of neuroblastoma X glioma hybrid.

PubMed

Ogura, A; Amano, T

1983-01-10

Mouse neuroblastoma clone N1E-115 cells and rat glioma clone C6 cells were hybridized and two new clones were isolated. One clone, designated NG115-301, possessed weak electric excitability to an applied current pulse, while another clone, NG115-401, generated an action potential in response to the pulse. The former clone responded to serotonin and catecholamines with slow hyperpolarizations, while the latter clone responded to catecholamines with transient depolarizations. Both clones did not respond to acetylcholine. These types of responses have not been reported in any available clones. These clones may enrich the repertoire of cell clones useful for the characterization of transmitter reception mechanisms in the nervous system.

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture

PubMed Central

Dempsey, Kate E; Riggio, Marcello P; Lennon, Alan; Hannah, Victoria E; Ramage, Gordon; Allan, David; Bagg, Jeremy

2007-01-01

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones analysed. In all, 28 phylotypes were identified: Lysobacter enzymogenes was the most abundant phylotype (31.4%), followed by Lysobacter sp. C3 (28.3%), gamma proteobacterium N4-7 (6.6%), Methylobacterium SM4 (4.7%) and Staphylococcus epidermidis (4.7%); 36 clones (7.0%) represented uncultivable phylotypes. We conclude that a diverse range of bacterial species are found within biofilms on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties. PMID:17501992

[Identification of antler powder components based on DNA barcoding technology].

PubMed

Jia, Jing; Shi, Lin-chun; Xu, Zhi-chao; Xin, Tian-yi; Song, Jing-yuan; Chen Shi, Lin

2015-10-01

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

Egg donation, surrogate mothering, and cloning: attitudes of men and women in Germany based on a representative survey.

PubMed

Stöbel-Richter, Yve; Goldschmidt, Susanne; Brähler, Elmar; Weidner, Kerstin; Beutel, Manfred

2009-07-01

To determine opinions and attitudes of the German general population toward the treatment methods of reproductive medicine: egg donation, surrogate mothering, and reproductive cloning. Representative survey. German general population: face-to-face interviews at home with 2,110 persons, aged 18-50 years. Patients were not included. No interventions took place. Approval and disapproval of treatment methods of reproductive medicine and preimplantation genetic diagnosis were assessed by questionnaires regarding medical, age, reasons, or general. Overall, the diverse treatment methods of reproductive medicine found comparable rates of approval and disapproval. Legalization of egg donation was approved by a slight majority (50.8%), particularly for medical reasons (35.9%). Surrogate mothering found lower overall rates of approval (43.7%), 28.5% supported an admission for medical reasons. Reproductive cloning was rejected by the vast majority (82.9%). Attitudes to reproductive medicine were affected by age and the individual reproductive experiences. New techniques in reproductive medicine and their development provide hope and health promises for affected couples but also entail long-term risks and ethical issues. Balancing the individual's right to a reproductive autonomy and choice and ethical standards will constitute a future challenge for society. Results demonstrate considerable uncertainty and information deficits in the community.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

An 'instant gene bank' method for gene cloning by mutant complementation.

PubMed

Gems, D; Aleksenko, A; Belenky, L; Robertson, S; Ramsden, M; Vinetski, Y; Clutterbuck, A J

1994-02-01

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Kuetemeyer, Kai; Lucas-Hahn, Andrea; Petersen, Bjoern; Lemme, Erika; Hassel, Petra; Niemann, Heiner; Heisterkamp, Alexander

2010-07-01

Since the birth of ``Dolly'' as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

[Microbial community in the Anammox process of thermal denitration tail liquid].

PubMed

Li, Jin; Yu, Deshuang; Zhao, Dan; Wang, Xiaochen

2014-12-01

An anaerobic sequencing batch reactor (ASBR) was used to treat thermal denitration tail liquid and microbial community was studied. Activated sludge was taken from the reactor for scanning electron microscope analysis. The images showed that the dominant cells in the flora were oval cocci. Its diameter was about 0.7 μm. Through a series of molecular biology methods such as extracting total DNA from the sludge, PCR amplification, positive clone authentication and sequencing, we obtained the 16S rDNA sequences of the flora. Phylogenetic tree and clone library were established. The universal bacteria primers of 27F-1492R PCR amplification system obtained 85 clones and could be divided into 21 OTUS. The proportions were as follows: Proteobacteria 61.18%; Acidobacteria 17.65%; Chlorobi 8.24%; Chlorofexi 5.88%; Gemmatimonadetes 3.53%; Nitrospirae 2.35% and Planctomycetes 1.18%. The specific anammox bacterial primers of pla46rc-630r and AMX368-AMX820 PCR amplification system obtained 45 clones. They were divided into 3 OTUS. Candidatus brocadia sp. occupied 95.6% and unknown strains occupied 4.4%.

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

PubMed

Koike, Tasuku; Wakai, Takuya; Jincho, Yuko; Sakashita, Akihiko; Kobayashi, Hisato; Mizutani, Eiji; Wakayama, Sayaka; Miura, Fumihito; Ito, Takashi; Kono, Tomohiro

2016-06-01

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier. © 2016 by the Society for the Study of Reproduction, Inc.

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

PubMed

Zou, Xiao-Hui; Bi, Zhi-Xiang; Guo, Xiao-Juan; Zhang, Zun; Zhao, Yang; Wang, Min; Zhu, Ya-Lu; Jie, Hong-Ying; Yu, Yang; Hung, Tao; Lu, Zhuo-Zhuang

2018-07-01

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics. Copyright © 2018 Elsevier B.V. All rights reserved.

Chromosome specific repetitive DNA sequences

DOEpatents

Moyzis, Robert K.; Meyne, Julianne

1991-01-01

A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

Clonal multiplication of Cymbidiums through tissue culture of the shoot meristem

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wimber, Donald E.

The propagation of clonal varieties of some orchids is at times exasperatingly slow and occasionally an almost futile effort. Clonal multiplication is generally confined to dlvidlng mature plants and to starting plants from pseudobulbs. There is, of course, the specialized technique for obtaining Phalaenopsis plantlets from the aseptic culture of inflorescence nodes, but this is basically the same thing as propagating plants from pseudobulbs. In certain cases it is highly desirable to rapidly multiply certain clones of orchids. Awarded varieties could thereby be dispersed with great rapidity where now it may take decades for some clones to became fairly common.more » Commercial flower production would be very much enhanced if certain desirable clones could be multiplied ad infinitum within a short time. Orchid flower production could then be placed more on a par with many of the other cut flowers and the clonal peculiarities of some fo the current hybrids could be pampered instead of ignored. This paper describes a tissue culture method for the rapid propagation of Cymbidium clones.« less

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion

PubMed Central

Yung, Pui Yi; Burke, Catherine; Lewis, Matt; Egan, Suhelen; Kjelleberg, Staffan; Thomas, Torsten

2009-01-01

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information. PMID:19767618

Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system.

PubMed

Lee, Wonyou; Song, Kilyoung; Lee, Inhyung; Shin, Hyungdo; Lee, Byeong Chun; Yeon, Seongchan; Jang, Goo

2015-01-01

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology.

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

PubMed

Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

2015-10-29

Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J.

When potato plants (Solanum tuberosum L. cv Norland) are subjected to oxone stress ethylene is emitted. Increases in ethylene production are often the result of increased expression of the enzyme ACC synthase. We used the polymerase chain reaction (PCR) to clone a cDNA encoding an ozone-induced ACC synthase. After treating potato plants with 300 ppb ozone for 4 h, RNA was extracted using a guanidinium isothiocyanate method. Using degenerate oligonucleotides corresponding to several conserved regions of ACC synthase sequences reported from different plant tissues as primers, we were able to reverse transcribe the RNA and amplify a cDNA for ACCmore » synthase. The clone is 1098 bp in length encoding for 386 amino acids comprising [approximately]80% of the protein. Computer analysis of the deduced amino acid sequence showed that our clone is 50-70% homologous with ACC synthase genes cloned from other plant tissues. Using the cDNA as a probe in northern analysis we found that there is little or no expression in control tissue: however there is a large increase in the expression of the ACC synthase message in response to ozone treatment.« less

(η(6)-Benzene)(carbonato-κ(2) O,O')[di-cyclohex-yl(naphthalen-1-ylmeth-yl)phosphane-κP]ruthenium(II) chloro-form tris-olvate.

PubMed

Gowrisankar, Saravanan; Neumann, Helfried; Spannenberg, Anke; Beller, Matthias

2014-07-01

The title compound, [Ru(CO3)(η(6)-C6H6){(C6H11)2P(CH2C10H7)}]·3CHCl3, was synthesized by carbonation of [RuCl2(η(6)-C6H6){(C6H11)2P(CH2C10H7)}] with NaHCO3 in methanol at room temperature. The Ru(II) atom is surrounded by a benzene ligand, a chelating carbonate group and a phosphane ligand in a piano-stool configuration. The crystal packing is consolidated by C-H⋯O and C-H⋯Cl hydrogen-bonding inter-actions between adjacent metal complexes and between the complexes and the solvent mol-ecules. The asymmetric unit contains one metal complex and three chloro-form solvent mol-ecules of which only one was modelled. The estimated diffraction contributions of the other two strongly disordered chloro-form solvent mol-ecules were substracted from the observed diffraction data using the SQUEEZE procedure in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148-155].

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

The development and characterisation of a bacterial artificial chromosome library for Fragaria vesca

PubMed Central

Bonet, Julio; Girona, Elena Lopez; Sargent, Daniel J; Muñoz-Torres, Monica C; Monfort, Amparo; Abbott, Albert G; Arús, Pere; Simpson, David W; Davik, Jahn

2009-01-01

Background The cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library. Methods A BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN). Findings Clones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size. Conclusion This BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR. PMID:19772672

Clone DB: an integrated NCBI resource for clone-associated data

PubMed Central

Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

2013-01-01

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

PubMed

Gerbod, D; Edgcomb, V P; Noël, C; Delgado-Viscogliosi, P; Viscogliosi, E

2000-09-01

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

Ab initio multiple cloning simulations of pyrrole photodissociation: TKER spectra and velocity map imaging

DOE PAGES

Makhov, Dmitry V.; Saita, Kenichiro; Martinez, Todd J.; ...

2014-12-11

In this study, we report a detailed computational simulation of the photodissociation of pyrrole using the ab initio Multiple Cloning (AIMC) method implemented within MOLPRO. The efficiency of the AIMC implementation, employing train basis sets, linear approximation for matrix elements, and Ehrenfest configuration cloning, allows us to accumulate significant statistics. We calculate and analyze the total kinetic energy release (TKER) spectrum and Velocity Map Imaging (VMI) of pyrrole and compare the results directly with experimental measurements. Both the TKER spectrum and the structure of the velocity map image (VMI) are well reproduced. Previously, it has been assumed that the isotropicmore » component of the VMI arises from long time statistical dissociation. Instead, our simulations suggest that ultrafast dynamics contributes significantly to both low and high energy portions of the TKER spectrum.« less

Ab initio multiple cloning simulations of pyrrole photodissociation: TKER spectra and velocity map imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makhov, Dmitry V.; Saita, Kenichiro; Martinez, Todd J.

In this study, we report a detailed computational simulation of the photodissociation of pyrrole using the ab initio Multiple Cloning (AIMC) method implemented within MOLPRO. The efficiency of the AIMC implementation, employing train basis sets, linear approximation for matrix elements, and Ehrenfest configuration cloning, allows us to accumulate significant statistics. We calculate and analyze the total kinetic energy release (TKER) spectrum and Velocity Map Imaging (VMI) of pyrrole and compare the results directly with experimental measurements. Both the TKER spectrum and the structure of the velocity map image (VMI) are well reproduced. Previously, it has been assumed that the isotropicmore » component of the VMI arises from long time statistical dissociation. Instead, our simulations suggest that ultrafast dynamics contributes significantly to both low and high energy portions of the TKER spectrum.« less

Rapid amplification of 5' complementary DNA ends (5' RACE).

PubMed

2005-08-01

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

PubMed

Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

2006-07-13

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies.

PubMed

Nishida, I; Sugiura, M; Enju, A; Nakamura, M

2000-12-01

A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

PubMed

Dehghan, Esmaeil; Reed, Darwin W; Covello, Patrick S; Hasanpour, Zeinab; Palazon, Javier; Oksman-Caldentey, Kirsi-Marja; Ahmadi, Farajollah Shahriari

2017-10-01

Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Cloning: A Review on Bioethics, Legal, Jurisprudence and Regenerative Issues in Iran.

PubMed

Nabavizadeh, Seyedeh Leila; Mehrabani, Davood; Vahedi, Zabihallah; Manafi, Farzad

2016-09-01

In recent years, the cloning technology has remarkably developed in Iran, but unfortunately, the required legal framework has not been created to support and protect such developments yet. This legal gap may lead to abuse of scientific researches to obtain illegal benefits and to undermine the intellectual property rights of scientists and researchers. Thus to prevent such consequences, the attempts should be made to create an appropriate legal-ethical system and an approved comprehensive law. In this review we concluded that the right method is guiding and controlling the cloning technology and banning the technique is not always fruitful. Of course, it should be taken into accounts that all are possible if the religion orders human cloning in the view of jurisprudence and is considered as permission. In other words, although the religious order on human cloning can be an absolute permission based on the strong principle of permission, it is not unlikely that in the future, corruption is proved to be real for them, Jurists rule it as secondary sanctity and even as primary one. If it is proved, the phenomenon is considered as example of required affairs based on creation of ethical, social and medical disorders, religious and ethical rulings cannot be as permission for it, and it seems that it is a point that only one case can be a response to it and it needs nothing but time.

Cloning: A Review on Bioethics, Legal, Jurisprudence and Regenerative Issues in Iran

PubMed Central

Nabavizadeh, Seyedeh Leila; Mehrabani, Davood; Vahedi, Zabihallah; Manafi, Farzad

2016-01-01

In recent years, the cloning technology has remarkably developed in Iran, but unfortunately, the required legal framework has not been created to support and protect such developments yet. This legal gap may lead to abuse of scientific researches to obtain illegal benefits and to undermine the intellectual property rights of scientists and researchers. Thus to prevent such consequences, the attempts should be made to create an appropriate legal-ethical system and an approved comprehensive law. In this review we concluded that the right method is guiding and controlling the cloning technology and banning the technique is not always fruitful. Of course, it should be taken into accounts that all are possible if the religion orders human cloning in the view of jurisprudence and is considered as permission. In other words, although the religious order on human cloning can be an absolute permission based on the strong principle of permission, it is not unlikely that in the future, corruption is proved to be real for them, Jurists rule it as secondary sanctity and even as primary one. If it is proved, the phenomenon is considered as example of required affairs based on creation of ethical, social and medical disorders, religious and ethical rulings cannot be as permission for it, and it seems that it is a point that only one case can be a response to it and it needs nothing but time. PMID:27853684

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography.

PubMed

Jacinto, R C; Gomes, B P F A; Desai, M; Rajendram, D; Shah, H N

2007-12-01

The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high-performance liquid chromatography. Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high-performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. All samples were positive for the presence of bacteria and a range of 7-13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

PubMed

Lanza, R P; Cibelli, J B; Diaz, F; Moraes, C T; Farin, P W; Farin, C E; Hammer, C J; West, M D; Damiani, P

2000-01-01

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Resistance to a bacterial parasite in the crustacean Daphnia magna shows Mendelian segregation with dominance

PubMed Central

Luijckx, P; Fienberg, H; Duneau, D; Ebert, D

2012-01-01

The influence of host and parasite genetic background on infection outcome is a topic of great interest because of its pertinence to theoretical issues in evolutionary biology. In the present study, we use a classical genetics approach to examine the mode of inheritance of infection outcome in the crustacean Daphnia magna when exposed to the bacterial parasite Pasteuria ramosa. In contrast to previous studies in this system, we use a clone of P. ramosa, not field isolates, which allows for a more definitive interpretation of results. We test parental, F1, F2, backcross and selfed parental clones (total 284 genotypes) for susceptibility against a clone of P. ramosa using two different methods, infection trials and the recently developed attachment test. We find that D. magna clones reliably exhibit either complete resistance or complete susceptibility to P. ramosa clone C1 and that resistance is dominant, and inherited in a pattern consistent with Mendelian segregation of a single-locus with two alleles. The finding of a single host locus controlling susceptibility to P. ramosa suggests that the previously observed genotype–genotype interactions in this system have a simple genetic basis. This has important implications for the outcome of host–parasite co-evolution. Our results add to the growing body of evidence that resistance to parasites in invertebrates is mostly coded by one or few loci with dominance. PMID:22167056

Resistance to a bacterial parasite in the crustacean Daphnia magna shows Mendelian segregation with dominance.

PubMed

Luijckx, P; Fienberg, H; Duneau, D; Ebert, D

2012-05-01

The influence of host and parasite genetic background on infection outcome is a topic of great interest because of its pertinence to theoretical issues in evolutionary biology. In the present study, we use a classical genetics approach to examine the mode of inheritance of infection outcome in the crustacean Daphnia magna when exposed to the bacterial parasite Pasteuria ramosa. In contrast to previous studies in this system, we use a clone of P. ramosa, not field isolates, which allows for a more definitive interpretation of results. We test parental, F1, F2, backcross and selfed parental clones (total 284 genotypes) for susceptibility against a clone of P. ramosa using two different methods, infection trials and the recently developed attachment test. We find that D. magna clones reliably exhibit either complete resistance or complete susceptibility to P. ramosa clone C1 and that resistance is dominant, and inherited in a pattern consistent with Mendelian segregation of a single-locus with two alleles. The finding of a single host locus controlling susceptibility to P. ramosa suggests that the previously observed genotype-genotype interactions in this system have a simple genetic basis. This has important implications for the outcome of host-parasite co-evolution. Our results add to the growing body of evidence that resistance to parasites in invertebrates is mostly coded by one or few loci with dominance.

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle.

PubMed

Yamanaka, Ken-Ichi; Kaneda, Masahiro; Inaba, Yasushi; Saito, Koji; Kubota, Kaiyu; Sakatani, Miki; Sugimura, Satoshi; Imai, Kei; Watanabe, Shinya; Takahashi, Masashi

2011-08-01

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

Analysis of intracellular cytokines using flowcytometry.

PubMed

Arora, Sunil K

2002-01-01

Characterization of T-cell clones and identification of functional subsets of the helper T-cells with polarized cytokine production is based on testing of cytokine expression. Several methods have been developed that allow cytokine expression to be measured like ELISA, RT-PCR, ELISPOT, ISH and flowcytometry. Among all these methods, monitoring of cytokine production using flowcytometric analysis has its own advantages and disadvantages. Multi-parametric characterization of cytokine production on single cell basis, without long-term culture and cloning along with high throughput of samples is main feature attached to flowcytometric analysis. The interpretation may be difficult at times due to change in the phenotype of the cells. Cells with similar surface phenotype but synthesizing different cytokines and having different functional characteristics can be analyzed with this technique.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

PubMed

Xu, Y L; Li, L; Wu, K; Peeters, A J; Gage, D A; Zeevaart, J A

1995-07-03

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yun-Ling; Li, Li; Wu, Keqiang

1995-07-03

The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidasemore » gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6 figs.« less

Cytotoxic Th1 and Th17 cells infiltrate the intestinal mucosa of Behcet patients and exhibit high levels of TNF-α in early phases of the disease

PubMed Central

Emmi, Giacomo; Silvestri, Elena; Bella, Chiara Della; Grassi, Alessia; Benagiano, Marisa; Cianchi, Fabio; Squatrito, Danilo; Cantarini, Luca; Emmi, Lorenzo; Selmi, Carlo; Prisco, Domenico; D’Elios, Mario Milco

2016-01-01

Abstract Background: Gastrointestinal involvement is one of the most serious in Behçet disease, potentially leading to severe complications. Aim of this study was to investigate at mucosal level the T-cell responses in Behçet patients with early intestinal involvement. Methods: We isolated T cells from intestinal mucosa of 8 patients with intestinal symptoms started within 6 months. T lymphocytes were cloned and analyzed for surface phenotype and cytokines production. Results: We obtained 382 T-cell clones: 324 were CD4+ and 58 were CD8+. Within the 324 CD4+ clones, 195 were able to secrete IFN-γ and TNF-α, but not IL-4, nor IL-17 thus showing a polarized Th1 profile, whereas CD4 clones producing both IFN-γ and IL-17 (Th1/Th17 profile) were 79. Likewise, the number of CD8 clones producing type 1 cytokines was higher than those of CD8 clones producing both type 1 and 2 cytokines. Almost all intestinal-derived T-cell clones expressed perforin-mediated cytotoxicity and Fas–Fas Ligand-mediated pro-apoptotic activity. Conclusions: Our results indicate that in the early stages of the disease, both Th1 and Th17 cells drive inflammation leading to mucosal damage via abnormal and long-lasting cytokines production as well as via both perforin- and Fas–Fas ligand-mediated cytotoxicity. Finally, all the T cells at mucosal level were able to produce large amount of TNF-α, suggesting that its production is a property of intestinal T cells of patients with early active intestinal disease. These results support the therapy with anti-TNF-α agents and suggest the use of anti-IL-17 monoclonal antibodies in Behçet patients with early intestinal involvement. PMID:27930541

Geotemporal Analysis of Neisseria meningitidis Clones in the United States: 2000–2005

PubMed Central

Wiringa, Ann E.; Shutt, Kathleen A.; Marsh, Jane W.; Cohn, Amanda C.; Messonnier, Nancy E.; Zansky, Shelley M.; Petit, Susan; Farley, Monica M.; Gershman, Ken; Lynfield, Ruth; Reingold, Arthur; Schaffner, William; Thompson, Jamie; Brown, Shawn T.; Lee, Bruce Y.; Harrison, Lee H.

2013-01-01

Background The detection of meningococcal outbreaks relies on serogrouping and epidemiologic definitions. Advances in molecular epidemiology have improved the ability to distinguish unique Neisseria meningitidis strains, enabling the classification of isolates into clones. Around 98% of meningococcal cases in the United States are believed to be sporadic. Methods Meningococcal isolates from 9 Active Bacterial Core surveillance sites throughout the United States from 2000 through 2005 were classified according to serogroup, multilocus sequence typing, and outer membrane protein (porA, porB, and fetA) genotyping. Clones were defined as isolates that were indistinguishable according to this characterization. Case data were aggregated to the census tract level and all non-singleton clones were assessed for non-random spatial and temporal clustering using retrospective space-time analyses with a discrete Poisson probability model. Results Among 1,062 geocoded cases with available isolates, 438 unique clones were identified, 78 of which had ≥2 isolates. 702 cases were attributable to non-singleton clones, accounting for 66.0% of all geocoded cases. 32 statistically significant clusters comprised of 107 cases (10.1% of all geocoded cases) were identified. Clusters had the following attributes: included 2 to 11 cases; 1 day to 33 months duration; radius of 0 to 61.7 km; and attack rate of 0.7 to 57.8 cases per 100,000 population. Serogroups represented among the clusters were: B (n = 12 clusters, 45 cases), C (n = 11 clusters, 27 cases), and Y (n = 9 clusters, 35 cases); 20 clusters (62.5%) were caused by serogroups represented in meningococcal vaccines that are commercially available in the United States. Conclusions Around 10% of meningococcal disease cases in the U.S. could be assigned to a geotemporal cluster. Molecular characterization of isolates, combined with geotemporal analysis, is a useful tool for understanding the spread of virulent meningococcal clones and patterns of transmission in populations. PMID:24349182

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos.

PubMed

Wakayama, Sayaka; Cibelli, Jose B; Wakayama, Teruhiko

2003-01-01

Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr(2+)-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

PubMed Central

Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

2009-01-01

Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an important step towards the identification of all genes in the citrus genome. Furthermore, public availability of the cDNA clones generated in this study, and not only their sequence, enables testing of the biological function of the genes represented in the collection. Expression of the citrus SEP3 homologue, CitrSEP, in Arabidopsis results in early flowering, along with other phenotypes resembling the over-expression of the Arabidopsis SEPALLATA genes. Our findings suggest that the members of the SEP gene family play similar roles in these quite distant plant species. PMID:19747386

Fundamental resource-allocating model in colleges and universities based on Immune Clone Algorithms

NASA Astrophysics Data System (ADS)

Ye, Mengdie

2017-05-01

In this thesis we will seek the combination of antibodies and antigens converted from the optimal course arrangement and make an analogy with Immune Clone Algorithms. According to the character of the Algorithms, we apply clone, clone gene and clone selection to arrange courses. Clone operator can combine evolutionary search and random search, global search and local search. By cloning and clone mutating candidate solutions, we can find the global optimal solution quickly.

Defense Against Chip Cloning Attacks Based on Fractional Hopfield Neural Networks.

PubMed

Pu, Yi-Fei; Yi, Zhang; Zhou, Ji-Liu

2017-06-01

This paper presents a state-of-the-art application of fractional hopfield neural networks (FHNNs) to defend against chip cloning attacks, and provides insight into the reason that the proposed method is superior to physically unclonable functions (PUFs). In the past decade, PUFs have been evolving as one of the best types of hardware security. However, the development of the PUFs has been somewhat limited by its implementation cost, its temperature variation effect, its electromagnetic interference effect, the amount of entropy in it, etc. Therefore, it is imperative to discover, through promising mathematical methods and physical modules, some novel mechanisms to overcome the aforementioned weaknesses of the PUFs. Motivated by this need, in this paper, we propose applying the FHNNs to defend against chip cloning attacks. At first, we implement the arbitrary-order fractor of a FHNN. Secondly, we describe the implementation cost of the FHNNs. Thirdly, we propose the achievement of the constant-order performance of a FHNN when ambient temperature varies. Fourthly, we analyze the electrical performance stability of the FHNNs under electromagnetic disturbance conditions. Fifthly, we study the amount of entropy of the FHNNs. Lastly, we perform experiments to analyze the pass-band width of the fractor of an arbitrary-order FHNN and the defense against chip cloning attacks capability of the FHNNs. In particular, the capabilities of defense against chip cloning attacks, anti-electromagnetic interference, and anti-temperature variation of a FHNN are illustrated experimentally in detail. Some significant advantages of the FHNNs are that their implementation cost is considerably lower than that of the PUFs, their electrical performance is much more stable than that of the PUFs under different temperature conditions, their electrical performance stability of the FHNNs under electromagnetic disturbance conditions is much more robust than that of the PUFs, and their amount of entropy is significantly higher than that of the PUFs with the same rank circuit scale.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis).

PubMed

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis)

PubMed Central

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated. PMID:27736862

Human cloning 2001.

PubMed

Healy, David L; Weston, Gareth; Pera, Martin F; Rombauts, Luk; Trounson, Alan O

2002-05-01

This review summaries human cloning from a clinical perspective. Natural human clones, that is, monozygotic twins, are increasing in the general community. Iatrogenic human clones have been produced for decades in infertile couples given fertility treatment such as ovulation induction. A clear distinction must be made between therapeutic cloning using embryonic stem cells and reproductive cloning attempts. Unlike the early clinical years of in vitro fertilization, with cloning there is no animal model that is safe and dependable. Until there is such a model, 'Dolly'-style human cloning is medically unacceptable.

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

ADAMTS13 Autoantibodies Cloned from Patients with Acquired Thrombotic Thrombocytopenic Purpura: 1. Structural and functional characterization in vitro

PubMed Central

Ostertag, Eric M.; Kacir, Stephen; Thiboutot, Michelle; Gulendran, Gayathri; Zheng, X. Long; Cines, Douglas B.; Siegel, Don L.

2016-01-01

BACKGROUND Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand Factor cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular/genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal IgG in patient plasma. RESULTS Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed to both the amino terminal domains and those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were non-inhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies. PMID:27040144

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

PubMed

Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

2018-01-26

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Fluoroquinolone-Resistant Enteric Bacteria in Sub-Saharan Africa: Clones, Implications and Research Needs

PubMed Central

Chattaway, Marie A.; Aboderin, Aaron O.; Fashae, Kayode; Okoro, Chinyere K.; Opintan, Japheth A.; Okeke, Iruka N.

2016-01-01

Fluoroquinolones came into widespread use in African countries in the early 2000s, after patents for the first generation of these drugs expired. By that time, quinolone antibacterial agents had been used intensively worldwide and resistant lineages of many bacterial species had evolved. We sought to understand which Gram negative enteric pandemic lineages have been reported from Africa, as well as the nature and transmission of any indigenous resistant clones. A systematic review of articles indexed in the Medline and AJOL literature databases was conducted. We report on the findings of 43 eligible studies documenting local or pandemic fluoroquinolone-resistant enteric clones in sub-Sahara African countries. Most reports are of invasive non-typhoidal Salmonella and Escherichia coli lineages and there have been three reports of cholera outbreaks caused by fluoroquinolone-resistant Vibrio cholerae O1. Fluoroquinolone-resistant clones have also been reported from commensals and animal isolates but there are few data for non-Enterobacteriaceae and almost none for difficult-to-culture Campylobacter spp. Fluoroquinolone-resistant lineages identified in African countries were universally resistant to multiple other classes of antibacterial agents. Although as many as 972 non-duplicate articles refer to fluoroquinolone resistance in enteric bacteria from Africa, most do not report on subtypes and therefore information on the epidemiology of fluoroquinolone-resistant clones is available from only a handful of countries in the subcontinent. When resistance is reported, resistance mechanisms and lineage information is rarely investigated. Insufficient attention has been given to molecular and sequence-based methods necessary for identifying and tracking resistant clones in Africa and more research is needed in this area. PMID:27148238

Biochemical, genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.

PubMed Central

Brenner, D J; Mayer, L W; Carlone, G M; Harrison, L H; Bibb, W F; Brandileone, M C; Sottnek, F O; Irino, K; Reeves, M W; Swenson, J M

1988-01-01

Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim. Images PMID:3262623

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

PubMed Central

Berdien, Belinda; Reinhard, Henrike; Meyer, Sabrina; Spöck, Stefanie; Kröger, Nicolaus; Atanackovic, Djordje; Fehse, Boris

2013-01-01

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies. PMID:23428899

Endolithic diversity of microorganisms on sandstone and implications for biogenic weathering

NASA Astrophysics Data System (ADS)

Hallmann, C.; Friedenberger, H.; Hoppert, M.

2012-04-01

Molecular methods allow a comprehensive view on uncultured microbial communities in dimension stone. In the presented study, we focus on depth profiles of microbial colonization in sandstones with different porosity and overall durability. All sandstones were taken from quarries where they were exposed to the environment for several years. Approximately 0.1 g of material from the stone surface, from 5 mm and from 30 mm depths was taken under sterile conditions and subjected to analysis of microbial DNA and culturing experiments. In particular, DNA was extracted from the material, the phylogenetic marker gene of eukaryotic organisms (18S rDNA) was amplified and used for generation of clone libraries, which were then analysed by sequencing. "Roter Wesersandstein" was just colonized at the material surface, predominantly with algal and fungal microorganisms. No environmental DNA could be isolated from depth profiles. From "Nebraer Sandstein" with high pore size (shown by thin sections), environmental DNA from depths down to 3 cm could be retrieved. Though the uppermost layer is dominated by microalgae (as concluded from the retrieved clones), the percentage of algal clones from 5 mm and 30 mm depths drop to 10 % of all clones. There, apart from filamentous fungi, moss clones clearly dominate the microbial community. At a depth of 30 mm, 70-80 % of the retrieved clones match to various mosses (Bryophyta). Though mosses do not form layers on the stone surfaces, moss rhizoids or protonemata must be abundant as endoliths inside the stone material. It is reasonable to assume that the rhizoids may contribute to an increase in pore size by active penetration of the clastic material, even though colonization of the surface by mosses is not obvious. This feature may imply stronger impact of stone decay induced by endolithic growth of bryophytes than hitherto observed.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

PubMed

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

PubMed Central

Michaelsen, Astrid; Piñar, Guadalupe

2010-01-01

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities. PMID:20449583

Cloning of non-human primates: the road “less traveled by”

PubMed Central

SPARMAN, MICHELLE L.; TACHIBANA, MASAHITO; MITALIPOV, SHOUKHRAT M.

2011-01-01

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model. PMID:21404187

Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

PubMed Central

Desomer, Jan; Dhaese, Patrick; Montagu, Marc Van

1990-01-01

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. Images PMID:16348290

Cloning of non-human primates: the road "less traveled by".

PubMed

Sparman, Michelle L; Tachibana, Masahito; Mitalipov, Shoukhrat M

2010-01-01

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.

Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.

PubMed

Luo, Jing; Lu, Liaoxun; Gu, Yanrong; Huang, Rong; Gui, Lin; Li, Saichao; Qi, Xinhui; Zheng, Wenping; Chao, Tianzhu; Zheng, Qianqian; Liang, Yinming; Zhang, Lichen

2018-06-07

Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

PubMed

Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

2013-01-01

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

PubMed Central

Sankian, Mojtaba; Vahedi, Fatemeh; Pazouki, Nazanin; Moghadam, Malihe; Jabbari Azad, Farahzad; Varasteh, Abdol-Reza

2012-01-01

Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen. PMID:26989705

Biodiversity hot spot on a hot spot: novel extremophile diversity in Hawaiian fumaroles.

PubMed

Wall, Kate; Cornell, Jennifer; Bizzoco, Richard W; Kelley, Scott T

2015-01-06

Fumaroles (steam vents) are the most common, yet least understood, microbial habitat in terrestrial geothermal settings. Long believed too extreme for life, recent advances in sample collection and DNA extraction methods have found that fumarole deposits and subsurface waters harbor a considerable diversity of viable microbes. In this study, we applied culture-independent molecular methods to explore fumarole deposit microbial assemblages in 15 different fumaroles in four geographic locations on the Big Island of Hawai'i. Just over half of the vents yielded sufficient high-quality DNA for the construction of 16S ribosomal RNA gene sequence clone libraries. The bacterial clone libraries contained sequences belonging to 11 recognized bacterial divisions and seven other division-level phylogenetic groups. Archaeal sequences were less numerous, but similarly diverse. The taxonomic composition among fumarole deposits was highly heterogeneous. Phylogenetic analysis found cloned fumarole sequences were related to microbes identified from a broad array of globally distributed ecotypes, including hot springs, terrestrial soils, and industrial waste sites. Our results suggest that fumarole deposits function as an "extremophile collector" and may be a hot spot of novel extremophile biodiversity. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing.

PubMed

Kim, Bong-Soo; Kim, Byung Kwon; Lee, Jae-Hak; Kim, Myungjin; Lim, Young Woon; Chun, Jongsik

2008-08-01

Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gamma-and Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which were further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

Physical Unclonable Function Hardware Keys Utilizing Kirchhoff-Law Secure Key Exchange and Noise-Based Logic

NASA Astrophysics Data System (ADS)

Kish, Laszlo B.; Kwan, Chiman

Weak unclonable function (PUF) encryption key means that the manufacturer of the hardware can clone the key but not anybody else. Strong unclonable function (PUF) encryption key means that even the manufacturer of the hardware is unable to clone the key. In this paper, first we introduce an "ultra" strong PUF with intrinsic dynamical randomness, which is not only unclonable but also gets renewed to an independent key (with fresh randomness) during each use via the unconditionally secure key exchange. The solution utilizes the Kirchhoff-law-Johnson-noise (KLJN) method for dynamical key renewal and a one-time-pad secure key for the challenge/response process. The secure key is stored in a flash memory on the chip to provide tamper-resistance and nonvolatile storage with zero power requirements in standby mode. Simplified PUF keys are shown: a strong PUF utilizing KLJN protocol during the first run and noise-based logic (NBL) hyperspace vector string verification method for the challenge/response during the rest of its life or until it is re-initialized. Finally, the simplest PUF utilizes NBL without KLJN thus it can be cloned by the manufacturer but not by anybody else.

Biodiversity hot spot on a hot spot: novel extremophile diversity in Hawaiian fumaroles

PubMed Central

Wall, Kate; Cornell, Jennifer; Bizzoco, Richard W; Kelley, Scott T

2015-01-01

Fumaroles (steam vents) are the most common, yet least understood, microbial habitat in terrestrial geothermal settings. Long believed too extreme for life, recent advances in sample collection and DNA extraction methods have found that fumarole deposits and subsurface waters harbor a considerable diversity of viable microbes. In this study, we applied culture-independent molecular methods to explore fumarole deposit microbial assemblages in 15 different fumaroles in four geographic locations on the Big Island of Hawai'i. Just over half of the vents yielded sufficient high-quality DNA for the construction of 16S ribosomal RNA gene sequence clone libraries. The bacterial clone libraries contained sequences belonging to 11 recognized bacterial divisions and seven other division-level phylogenetic groups. Archaeal sequences were less numerous, but similarly diverse. The taxonomic composition among fumarole deposits was highly heterogeneous. Phylogenetic analysis found cloned fumarole sequences were related to microbes identified from a broad array of globally distributed ecotypes, including hot springs, terrestrial soils, and industrial waste sites. Our results suggest that fumarole deposits function as an “extremophile collector” and may be a hot spot of novel extremophile biodiversity. PMID:25565172

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

PubMed

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences.

PubMed

Bricheux, G; Brugerolle, G

1997-08-01

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landry, L.G.; Pell, E.J.

Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH)more » groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.« less

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis.

PubMed

Geeta Vani, R; Varghese, C M; Rao, M R

1999-12-15

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process. Copyright 1999 Academic Press.

Enhancer scanning to locate regulatory regions in genomic loci

PubMed Central

Buckley, Melissa; Gjyshi, Anxhela; Mendoza-Fandiño, Gustavo; Baskin, Rebekah; Carvalho, Renato S.; Carvalho, Marcelo A.; Woods, Nicholas T.; Monteiro, Alvaro N.A.

2016-01-01

The present protocol provides a rapid, streamlined and scalable strategy to systematically scan genomic regions for the presence of transcriptional regulatory regions active in a specific cell type. It creates genomic tiles spanning a region of interest that are subsequently cloned by recombination into a luciferase reporter vector containing the Simian Virus 40 promoter. Tiling clones are transfected into specific cell types to test for the presence of transcriptional regulatory regions. The protocol includes testing of different SNP (single nucleotide polymorphism) alleles to determine their effect on regulatory activity. This procedure provides a systematic framework to identify candidate functional SNPs within a locus during functional analysis of genome-wide association studies. This protocol adapts and combines previous well-established molecular biology methods to provide a streamlined strategy, based on automated primer design and recombinational cloning to rapidly go from a genomic locus to a set of candidate functional SNPs in eight weeks. PMID:26658467

Measles virus–specific plasma cells are prominent in subacute sclerosing panencephalitis CSF

PubMed Central

Owens, G.P.; Ritchie, A.M.; Gilden, D.H.; Burgoon, M.P.; Becker, D.; Bennett, J.L.

2012-01-01

Objective To demonstrate the specificity of expanded CD138+ plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). Methods IgG variable region sequences of single-antibody-secreting CD138+ cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138+ clones and assayed for immunoreactivity against MV proteins. Results Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. Conclusions Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders. PMID:17515543

Evaluation of genetic divergence among clones of conilon coffee after scheduled cycle pruning.

PubMed

Dalcomo, J M; Vieira, H D; Ferreira, A; Lima, W L; Ferrão, R G; Fonseca, A F A; Ferrão, M A G; Partelli, F L

2015-11-30

Coffea canephora genotypes from the breeding program of Instituto Capixaba de Pesquisa e Extensão Rural were evaluated, and genetic diversity was estimated with the aim of future improvement strategies. From an initial group of 55 genotypes, 18 from the region of Castelo, ES, were selected, and three clones of the cultivars "Vitória" and "robusta tropical." Upon completion of the scheduled cycle pruning, 17 morphoagronomic traits were measured in the 22 genotypes selected. The principal components method was used to evaluate the contributions relative to the traits. The genetic dissimilarity matrix was obtained through Mahalanobis generalized distance, and genotypes were grouped using the hierarchical method based on the mean of the distances. The most promising clones of Avaliação Castelo were AC02, AC03, AC12, AC13, AC22, AC24, AC26, AC27, AC28, AC29, AC30, AC35, AC36, AC37, AC39, AC40, AC43, and AC46. These methods detected high genetic variability, grouping, by similarity, the genotypes in five groups. The trait that contributed the least to genetic divergence was the number of leaves in plagiotropic branches; however, this was not eliminated, because discarding it altered the groups. There are superior genotypes with potential for use in the next stages of the breeding program, aimed at both the composition of clonal variety and hybridizations.

Alimentary Tract Bacteria Isolated and Identified with API-20E and Molecular Cloning Techniques from Australian Tropical Fruit Flies, Bactrocera cacuminata and B. tryoni

PubMed Central

Thaochan, N.; Drew, R. A. I.; Hughes, J. M.; Vijaysegaran, S.; Chinajariyawong, A.

2010-01-01

Bacteria were isolated from the crop and midgut of field collected Bactrocera cacuminata (Hering) and Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Two methods were used, firstly isolation onto two types of bacteriological culture media (PYEA and TSA) and identification using the API-20E diagnostic kit, and secondly, analysis of samples using the 16S rRNA gene molecular diagnostic method. Using the API-20E method, 10 genera and 17 species of bacteria in the family Enterobacteriaceae were identified from cultures growing on the nutrient agar. The dominant species in both the crop and midgut were Citrobacter freundii, Enterobacter cloacae and Klebsiella oxytoca. Providencia rettgeri, Klebsiella pneumoniae ssp ozaenae and Serratia marcescens were isolated from B. tryoni only. Using the molecular cloning technique that is based on 16S rRNA gene sequences, five bacteria classes were dignosed — Alpha-, Beta-, Gamma- and Delta- Proteobacteria and Firmicutes — including five families, Leuconostocaceae, Enterococcaceae, Acetobacteriaceae, Comamonadaceae and Enterobacteriaceae. The bacteria affiliated with Firmicutes were found mainly in the crop while the Gammaproteobacteria, especially the family Enterobacteriaceae, was dominant in the midgut. This paper presents results from the first known application of molecular cloning techniques to study bacteria within tephritid species and the first record of Firmicutes bacteria in these flies. PMID:20883132

Mercury in water and biomass of microbial communities in hot springs of Yellowstone National Park, USA

USGS Publications Warehouse

King, S.A.; Behnke, S.; Slack, K.; Krabbenhoft, D.P.; Nordstrom, D. Kirk; Burr, M.D.; Striegl, Robert G.

2006-01-01

Ultra-clean sampling methods and approaches typically used in pristine environments were applied to quantify concentrations of Hg species in water and microbial biomass from hot springs of Yellowstone National Park, features that are geologically enriched with Hg. Microbial populations of chemically-diverse hot springs were also characterized using modern methods in molecular biology as the initial step toward ongoing work linking Hg speciation with microbial processes. Molecular methods (amplification of environmental DNA using 16S rDNA primers, cloning, denatured gradient gel electrophoresis (DGGE) screening of clone libraries, and sequencing of representative clones) were used to examine the dominant members of microbial communities in hot springs. Total Hg (THg), monomethylated Hg (MeHg), pH, temperature, and other parameters influential to Hg speciation and microbial ecology are reported for hot springs water and associated microbial mats. Several hot springs indicate the presence of MeHg in microbial mats with concentrations ranging from 1 to 10 ng g-1 (dry weight). Concentrations of THg in mats ranged from 4.9 to 120,000 ng g-1 (dry weight). Combined data from surveys of geothermal water, lakes, and streams show that aqueous THg concentrations range from l to 600 ng L-1. Species and concentrations of THg in mats and water vary significantly between hot springs, as do the microorganisms found at each site. ?? 2006.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system

PubMed Central

Lee, Wonyou; Song, Kilyoung; Lee, Inhyung; Shin, Hyungdo; Lee, Byeong Chun

2015-01-01

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology PMID:26119166

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

PubMed

Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

2015-05-25

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Prokaryotic phylogenetic diversity of Hungarian deep subsurface geothermal well waters.

PubMed

Németh, Andrea; Szirányi, Barbara; Krett, Gergely; Janurik, Endre; Kosáros, Tünde; Pekár, Ferenc; Márialigeti, Károly; Borsodi, Andrea K

2014-09-01

Geothermal wells characterized by thermal waters warmer than 30°C can be found in more than 65% of the area of Hungary. The examined thermal wells located nearby Szarvas are used for heating industrial and agricultural facilities because of their relatively high hydrocarbon content. The aim of this study was to reveal the prokaryotic community structure of the water of SZR18, K87 and SZR21 geothermal wells using molecular cloning methods and Denaturing Gradient Gel Electrophoresis (DGGE). Water samples from the outflow pipes were collected in 2012 and 2013. The phylogenetic distribution of archaeal molecular clones was very similar in each sample, the most abundant groups belonged to the genera Methanosaeta, Methanothermobacter and Thermofilum. In contrast, the distribution of bacterial molecular clones was very diverse. Many of them showed the closest sequence similarities to uncultured clone sequences from similar thermal environments. From the water of the SZR18 well, phylotypes closely related to genera Fictibacillus and Alicyclobacillus (Firmicutes) were only revealed, while the bacterial diversity of the K87 well water was much higher. Here, the members of the phyla Thermodesulfobacteria, Proteobacteria, Nitrospira, Chlorobi, OP1 and OPB7 were also detected besides Firmicutes.

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

PubMed Central

Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

2016-01-01

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

Male specific genes from dioecious white campion identified by fluorescent differential display.

PubMed

Scutt, Charles P; Jenkins, Tom; Furuya, Masaki; Gilmartin, Philip M

2002-05-01

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

PubMed

Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi

2017-11-01

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.

Enrichment and identification of cellulolytic bacteria from the gastrointestinal tract of Giant African snail, Achatina fulica.

PubMed

Pawar, Kiran D; Dar, Mudasir A; Rajput, Bharati P; Kulkarni, Girish J

2015-02-01

The cellulolytic bacterial community structure in gastrointestinal (GI) tract of Achatina fulica was studied using culture-independent and -dependent methods by enrichment in carboxymethyl cellulose (CMC). Culture-dependent method indicated that GI tract of snail was dominated by Enterobacteriaceae members. When tested for cellulase activities, all isolates obtained by culture-dependent method showed both or either of CMCase or avicelase activity. Isolate identified as Citrobacter freundii showed highest CMCase and medium avicelase activity. Sequencing of clones from the 16S rRNA gene clone library identified ten operational taxonomic units (OTUs), which were affiliated to Enterobacteriaceae of phylum Gammaproteobacteria. Of these ten OTUs, eight OTUs closely matched with Enterobacter and Klebsiella genera. The most abundant OTU allied to Klebsiella oxytoca accounted for 70 % of the total sequences. The members of Klebsiella and Enterobacter were observed by both methods indicating their dominance among the cellulolytic bacterial community in the GI tract of the snail.

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity▿

PubMed Central

Withers, Sydnor T.; Gottlieb, Shayin S.; Lieu, Bonny; Newman, Jack D.; Keasling, Jay D.

2007-01-01

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol. PMID:17693564

Human cloning: can it be made safe?

PubMed

Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian

2003-11-01

There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bartkiewicz, Karol; Miranowicz, Adam

We find an optimal quantum cloning machine, which clones qubits of arbitrary symmetrical distribution around the Bloch vector with the highest fidelity. The process is referred to as phase-independent cloning in contrast to the standard phase-covariant cloning for which an input qubit state is a priori better known. We assume that the information about the input state is encoded in an arbitrary axisymmetric distribution (phase function) on the Bloch sphere of the cloned qubits. We find analytical expressions describing the optimal cloning transformation and fidelity of the clones. As an illustration, we analyze cloning of qubit state described by themore » von Mises-Fisher and Brosseau distributions. Moreover, we show that the optimal phase-independent cloning machine can be implemented by modifying the mirror phase-covariant cloning machine for which quantum circuits are known.« less

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin

PubMed Central

Akbarzadeh-Sharbaf, Soudabeh; Yakhchali, Bagher; Minuchehr, Zarrin; Shokrgozar, Mohammad Ali; Zeinali, Sirous

2012-01-01

Background: There is a novel hypothesis in that antibodies may have specificity for two distinct antigens that have been named “dual specificity”. This hypothesis was evaluated for some defined therapeutic monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab, Bevacizumab, and Cetuximab. In silico design and construction of expression vectors for trastuzumab monoclonal antibody also in this work were performed. Materials and Methods: First, in bioinformatics studies the 3D structures of concerned mAbs were obtained from the Protein Data Bank (PDB). Three-dimensional structural alignments were performed with SIM and MUSTANG softwares. AutoDock4.2 software also was used for the docking analysis. Second, the suitable genes for trastuzumab heavy and light chains were designed, synthesized, and cloned in the prokaryotic vector. These fragments individually were PCR amplified and cloned into pcDNA™ 3.3-TOPO® and pOptiVEC™ TOPO® shuttle vectors, using standard methods. Results: First, many bioinformatics tools and softwares were applied but we did not meet any new dual specificity in the selected antibodies. In the following step, the suitable expression cascade for the heavy and light chains of Trastuzumab therapeutic mAb were designed and constructed. Gene cloning was successfully performed and created constructs were confirmed using gene mapping and sequencing. Conclusions: This study was based on a recently developed technology for mAb expression in mammalian cells. The obtained constructs could be successfully used for biosimilar recombinant mAb production in CHO DG44 dihydrofolate reductase (DHFR) gene deficient cell line in the suspension culture medium. PMID:23210080

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype

PubMed Central

Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette

2013-01-01

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype. PMID:23974297

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

PubMed

Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette

2013-01-01

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.

Distribution of quantum Fisher information in asymmetric cloning machines

PubMed Central

Xiao, Xing; Yao, Yao; Zhou, Lei-Ming; Wang, Xiaoguang

2014-01-01

An unknown quantum state cannot be copied and broadcast freely due to the no-cloning theorem. Approximate cloning schemes have been proposed to achieve the optimal cloning characterized by the maximal fidelity between the original and its copies. Here, from the perspective of quantum Fisher information (QFI), we investigate the distribution of QFI in asymmetric cloning machines which produce two nonidentical copies. As one might expect, improving the QFI of one copy results in decreasing the QFI of the other copy. It is perhaps also unsurprising that asymmetric phase-covariant cloning outperforms universal cloning in distributing QFI since a priori information of the input state has been utilized. However, interesting results appear when we compare the distributabilities of fidelity (which quantifies the full information of quantum states), and QFI (which only captures the information of relevant parameters) in asymmetric cloning machines. Unlike the results of fidelity, where the distributability of symmetric cloning is always optimal for any d-dimensional cloning, we find that any asymmetric cloning outperforms symmetric cloning on the distribution of QFI for d ≤ 18, whereas some but not all asymmetric cloning strategies could be worse than symmetric ones when d > 18. PMID:25484234

Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus).

PubMed

Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L

2010-12-01

For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our results suggest similar cognitive capacities of kin and non kin discrimination in AI and cloned animals. Kinship may be a common factor in determining the social grouping within a herd. Copyright © 2010 Elsevier Inc. All rights reserved.

Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes.

PubMed

Gao, Fan-Xiang; Wang, Yang; Zhang, Qi-Ya; Mou, Cheng-Yan; Li, Zhi; Deng, Yuan-Sheng; Zhou, Li; Gui, Jian-Fang

2017-07-24

Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A + , candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A + , F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A + and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A + . In contrast to strong immune defense in resistant clone H, susceptible clone A + showed remarkable up-regulation of genes related to apoptosis or death, indicating that clone A + failed to resist virus offensive and evidently induced apoptosis or death. Our study is the first attempt to screen distinct resistances and immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.

All God's Chillun Got a Mama and a Papa?

ERIC Educational Resources Information Center

Hollander, Willard F.

1972-01-01

Describes alternate methods of schematically representing genetic lines of descent, and illustrates the different patterns obtained with random mating, line breeding, parthanogentic males, cloning, and grafting. (AL)

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

PubMed

Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars; Stagsted, Jan; Boye, Mette

2013-02-07

Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

In vivo Elimination of Parental Clones in General and Site-directed Mutagenesis

PubMed Central

Holland, Erika G.; Acca, Felicity E.; Belanger, Kristina M.; Bylo, Mary E.; Kay, Brian K.; Weiner, Michael P.; Kiss, Margaret M.

2015-01-01

The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5’-CCGCGG-3’ and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 107 from a single transformation and with greater than 90% recombinant clones. PMID:25523926

In vivo elimination of parental clones in general and site-directed mutagenesis.

PubMed

Holland, Erika G; Acca, Felicity E; Belanger, Kristina M; Bylo, Mary E; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2015-02-01

The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones. Copyright © 2014 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamoureux, Louis-Philippe; Navez, Patrick; Cerf, Nicolas J.

It is shown that any quantum operation that perfectly clones the entanglement of all maximally entangled qubit pairs cannot preserve separability. This 'entanglement no-cloning' principle naturally suggests that some approximate cloning of entanglement is nevertheless allowed by quantum mechanics. We investigate a separability-preserving optimal cloning machine that duplicates all maximally entangled states of two qubits, resulting in 0.285 bits of entanglement per clone, while a local cloning machine only yields 0.060 bits of entanglement per clone.

Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

PubMed

Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

2011-10-01

Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Cloning: applications in humans 2. Ethical considerations].

PubMed

de Wert, G M; Geraedts, J P

2001-05-01

Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustify as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

[Cloning: applications in humans. II. Ethical considerations].

PubMed

de Wert, G M; Geraedts, J P

2000-05-13

Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustifiable as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

Harmonizing the international regulation of embryonic stem cell research: possibilities, promises and potential pitfalls.

PubMed

Campbell, Angela; Nycum, Gillian

2005-01-01

Despite near unanimous global opposition to human reproductive cloning, the United Nations has been unable to reach a consensus as to how cloning practices should be regulated at the international level. As a result, the U.N. objective of establishing binding international regulations governing cloning and stem cell research has yet to be achieved. Given the lack of consensus that exists within the global community on this topic, it seems that any attempt to harmonize the international regulation of cloning and stem cell science will face important obstacles. This paper seeks to illuminate the particular challenges to harmonizing international laws and policies related to stem cell research and human cloning, and to investigate potential methods for overcoming these challenges. By drawing on two other areas in which regulatory harmonization has been attempted, namely: environmental and human safety aspects of international trade, and pharmaceutical research and development, we study approaches to global regulatory harmonization. We conclude that while the challenges to harmonization are diverse and important, so too are the benefits of establishing uniformity in approaches to stem cell research worldwide. This paper proposes a model for harmonizing the regulation of stem cell research that focuses on broader norms and principles rather than specific rules. It further recommends that such harmonization should occur through a process initiated and developed by an independent international agency marked by diversity, both in terms of the cultural identities and perspectives represented, and the interdisciplinary expertise of its members.

Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Akbari, Bahman; Ahdi Khosroshahi, Shiva

2016-12-01

Purpose: EGFRvIII as the most common mutant variant of the epidermal growth factor receptor is resulting from deletion of exons 2-7 in the coding sequence and junction of exons 1 and 8 through a novel glycine residue. EGFRvIII is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. The aim of the present study was identification of a novel single chain antibody against EGFRvIII as a promising target for cancer therapy. Methods: In this study, a synthetic peptide corresponding to EGFRvIII protein was used for screening a naive human scFv phage library. A novel five-round selection strategy was used for enrichment of rare specific clones. Results: After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, only three clones had expected size in PCR reaction. The specific interaction of two of the scFv clones with EGFRvIII was confirmed by indirect ELISA. One phage clone with higher affinity in scFv ELISA was purified for further analysis. The purity of the produced scFv antibody was confirmed using SDS-PAGE and Western blotting analyses. Conclusion: In the present study, a human anti- EGFRvIII scFv with high affinity was first identified from a scFv phage library. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers.

Expression of recombinant human α-lactalbumin in milk of transgenic cloned pigs is sufficient to enhance intestinal growth and weight gain of suckling piglets.

PubMed

Ma, Jin; Li, Qiuyan; Li, Yan; Wen, Xiao; Li, Zhiyuan; Zhang, Zaihu; Zhang, Jiuming; Yu, Zhengquan; Li, Ning

2016-06-10

Human α-lactalbumin (HLA) has very high nutritional value and important physiological functions during the neonatal period. The peptides derived from HLA provide diverse health benefits including antimicrobial, antiviral, immune-modulating, and antihypertensive effects. Thus, it is worth investigating the effects on offspring development of increasing HLA in milk. In this study, we found that recombinant human α-lactalbumin (rHLA) exhibits efficient inhibition of dipeptidyl peptidase-IV (DPP-IV) activity in an in vitro simulated gastrointestinal digestion system. Using a BAC clone containing the complete HLA gene as a candidate vector, we generated two lines of transgenic cloned sows via somatic cell nuclear transfer that over-expressed rHLA. The average concentrations of rHLA in milk from the two lines of transgenic cloned sows were 2.24 ± 0.71 mg/ml and 2.67 ± 1.29 mg/ml. The feeding experiments revealed that rHLA represses dipeptidyl peptidase-IV (DPP-IV) activity in vivo. Furthermore, the piglets reared by rHLA transgenic cloned sows exhibit better performance in gain of body weight and intestine growth than the control piglets reared by non-transgenic sows. Therefore, these findings indicate that rHLA could serve as a natural precursor for a DPP-IV inhibitor, and the transgenic technology that produced the over-expression of rHLA could be a useful method for pig breeders to improve lactation performance. Copyright © 2016. Published by Elsevier B.V.

Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem.

PubMed

Oh, H J; Kim, M K; Jang, G; Kim, H J; Hong, S G; Park, J E; Park, K; Park, C; Sohn, S H; Kim, D Y; Shin, N S; Lee, B C

2008-09-01

The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

PubMed

Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

2016-09-01

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

PubMed

de Souza, Aracele M; de Araújo, Flávia C F; Fontes, Cor J F; Carvalho, Luzia H; de Brito, Cristiana F A; de Sousa, Taís N

2015-08-25

Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection. The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated. The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections. Depending on the type of marker used, a considerable amplification bias was observed, which may have serious implications for the characterization of the complexity of a P. vivax infection. Based on the performance of markers in artificial mixtures of DNA and natural infections, a minimum panel of four genetic markers (PvMS6, PvMS7, MN21, and msp1B10) was defined, and these markers are highly informative regarding the genetic variability of P. vivax populations.

Comparison of the X-radiation, drug and ultraviolet-radiation responses of clones isolated from a human colorectal tumor cell line.

PubMed

Qutob, Sami S; Multani, Asha S; Pathak, S; Feng, Y; Kendal, Wayne S; Ng, Cheng E

2004-03-01

We isolated several clones with a wide range of responses to X radiation from an unirradiated human colorectal (HCT 116) tumor cell line. The responses of one of these clones (HCT116-Clone10) and nine other clones to either fractionated or acute (i.e. single, nonfractionated doses) X irradiation in vitro was similar to that of the parental cell line. By contrast, after the same types of treatment, another clone (HCT116-Clone2) manifested a significantly increased survival whereas a third clone (HCT116-CloneK) manifested a significantly decreased survival relative to the parental cell line. This suggested that they were, respectively, a radioresistant and a radiosensitive clone. All three clones (clones 2, 10, K) retained their tumorigenic phenotype and formed tumors in nude mice. G-banding studies demonstrated that they were of human origin and were derived from the same parental cell line. The metaphases of HCT116-Clone2 demonstrated features commonly associated with genomic instability (i.e. mitotic catastrophe including chromosome and chromatid breaks, dicentrics and additional nonclonal markers). Data obtained by quantitative fluorescence in situ hybridization (Q- FISH) analysis failed to demonstrate any apparent correlation between the radiosensitivity and the relative telomere content of these three clones. Interestingly, HCT116-CloneK was the most resistant to several chemotherapeutic drugs (topotecan, camptothecin, etoposide and cisplatin) with diverse mechanisms of action. Also, there were no significant differences in the survivals of the three clones after treatment with UV radiation. Because of the lack of overlap among the relative sensitivities of these clones to X radiation, chemotherapeutic drugs and UV radiation, these clones may be useful models for evaluating the genetic basis of the response of human tumor cells to these treatment agents both in vitro and in vivo.

Ethical issues in animal cloning.

PubMed

Fiester, Autumn

2005-01-01

The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

PubMed

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.

PubMed

Liu, Xia; Li, Tuoping; Hart, Darren J; Gao, Song; Wang, Hongling; Gao, Herui; Xu, Shumin; Zhang, Yifeng; Liu, Yifei; An, Yingfeng

2018-03-18

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc. Copyright © 2018 Elsevier Inc. All rights reserved.

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server

PubMed Central

Ebhardt, H Alexander; Wiese, Kay C; Unrau, Peter J

2006-01-01

Background DNA sequencing is used ubiquitously: from deciphering genomes[1] to determining the primary sequence of small RNAs (smRNAs) [2-5]. The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for ~700 smRNA sequences[6]. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. Results Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project[6]. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from ~700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on Conclusion Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries [6-8].Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their annotation in a MySQL database, BlastN[9] comparison of new inserts to dynamic and static databases make it a powerful new tool in any laboratory using DNA sequencing. Ebbie also prevents manual mistakes during the excision process and speeds up annotation and data-entry. Once the server is installed locally, its access can be restricted to protect sensitive new DNA sequencing data. Ebbie was primarily designed for smRNA cloning projects, but can be applied to a variety of RNA and DNA cloning projects[2,3,10,11]. PMID:16584563

A journey through horse cloning.

PubMed

Gambini, Andrés; Maserati, Marc

2017-01-01

Interest in equine somatic cell nuclear transfer technology has increased significantly since the first equid clones were produced in 2003. This is demonstrated by the multiple commercial equine cloning companies having produced numerous cloned equids to date; worldwide, more than 370 cloned horses have been produced in at least six different countries. Equine cloning can be performed using several different approaches, each with different rates of success. In this review we cover the history and applications of equine cloning and summarise the major scientific advances in the development of this technology in horses. We explain the advantages and disadvantages of different procedures to produce cloned equine embryos and describe the current status of equine clone commercialisation, along with observations of differences in regional breed association registration regulations.

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

PubMed

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

2013-02-01

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs.

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

PubMed Central

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan

2013-01-01

Abstract Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150–199, 200–249, 250–299, 300–349, or 350–450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53±0.34) was similar with that associated with P,D,L,Y-FFBs (2.72±0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47±0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150–199 to 200–450 per recipient. However, increase of the number of transferred embryos from 200–249 to 250–450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250–450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200–249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs. PMID:23256540

[Offspring quality and its related factors of different Brachionus calyciflorus clones].

PubMed

Dong, Lili; Xi, Yilong; Zhang, Lei

2006-12-01

This paper studied the neonate starvation-endurance duration of four Brachionus calyciflorus clones (Clone A, B, C and D) with different biochemical-genetic characteristics at 15 degrees C, 20 degrees C, 25 degrees C and 30 degrees C, and the relationships of this duration with the temperature and the body- and egg volumes of B. calyciflorus. The results showed that at 15 degrees C, the neonates of Clone B had the shortest starvation-endurance duration (45.67 h); at 20 degrees C and 25 degrees C, the neonates' starvation-endurance duration of Clone C was the longest, being 61.33 h and 72.01 h, respectively; while at 30 degrees C, this duration of Clone A was the longest (40.11 h). The neonates' starvation-endurance duration of Clone A was the longest at 15 degrees C, those of Clone B and C were the shortest at 30 degrees C, while that of Clone D decreased with raising temperature. The neonates' starvation-endurance duration of all the four clones was negatively correlated with temperature. There was a negative correlation between this duration of Clone A and its egg volume, and the reverse was true for Clone C. The neonates' starvation-endurance duration of Clone B and D was positively correlated with the body volume of rotifer mother.

Challenge inoculations to test for Dutch elm disease tolerance: a summary of Methods used by various researchers

USDA-ARS?s Scientific Manuscript database

A variety of methods have been used by different research groups to “challenge” inoculate American elms (Ulmus americana) with the purpose of determining whether some clones may be resistant to the Dutch elm disease fungus. The methods used by seven research groups are described, along with observat...

The topsy-turvy cloning law.

PubMed

Brassington, Iain; Oultram, Stuart

2011-03-01

In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

PubMed

Lukan, Tjaša; Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

PubMed Central

Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. PMID:29300787

Mitochondrial DNA mutations in single human blood cells.

PubMed

Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

2015-09-01

Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. Copyright © 2015 Elsevier B.V. All rights reserved.

Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

PubMed Central

2011-01-01

Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures. PMID:22093809

Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Feifei; Gao, Feng; Li, Honglin

The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

Universal ligation-detection-reaction microarray applied for compost microbes

PubMed Central

Hultman, Jenni; Ritari, Jarmo; Romantschuk, Martin; Paulin, Lars; Auvinen, Petri

2008-01-01

Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR) based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS) area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities. PMID:19116002

Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

PubMed

Rahman, Muhammad H; Rajora, Om P

2002-12-01

Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same species showed higher microsatellite DNA similarities than the clones from the different species. A UPGMA cluster plot constructed from the microsatellite genotypic similarities separated the 96 clones into six major groups corresponding to their species. Populus nigra var. italica clones were genetically differentiated from the P. nigra var. nigra clones. Microsatellite DNA markers could be useful in genetic fingerprinting, identification, classification, certification, and registration of clones, clultivars, and varieties as well as genetic resource management and protection of plant breeders' rights in Populus.

Serial bull cloning by somatic cell nuclear transfer.

PubMed

Kubota, Chikara; Tian, X Cindy; Yang, Xiangzhong

2004-06-01

Although the list of species successfully cloned continues to grow, serial cloning has not been reported in species other than the mouse. Here we describe two live births of second-generation clones of a bull. Clones of the first and second generations appear healthy and have normal telomere lengths. Our attempts to produce the third generation of clones were unsuccessful.

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

PubMed

Kishigami, Satoshi; Bui, Hong-Thuy; Wakayama, Sayaka; Tokunaga, Kenzo; Van Thuan, Nguyen; Hikichi, Takafusa; Mizutani, Eiji; Ohta, Hiroshi; Suetsugu, Rinako; Sata, Tetsutaro; Wakayama, Teruhiko

2007-02-01

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun

Research highlights: {yields} In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. {yields} The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. {yields} The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. {yields} P53 status is not associated with the occurrence of unsensitized clone. {yields} Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeuticmore » regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC{sup -/-} cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC{sup -/-} clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.« less

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences†

PubMed Central

Speksnijder, Arjen G. C. L.; Kowalchuk, George A.; De Jong, Sander; Kline, Elizabeth; Stephen, John R.; Laanbroek, Hendrikus J.

2001-01-01

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries. PMID:11133483

U.S. consumers attitudes toward farm animal cloning.

PubMed

Brooks, Kathleen R; Lusk, Jayson L

2011-10-01

In January 2008, the United States Food and Drug Administration concluded "meat and milk from cattle, swine, and goat clones or their offspring are as safe to eat as food we eat from those species now" (U.S. FDA, 2010). However, cloning remains a very controversial topic. A web-based survey administered by Knowledge Networks was used to determine U.S. consumers' awareness of and attitudes toward meat and milk from cloned cattle. Findings reveal consumers do not differentiate much between products from cloned animals and products from non-cloned animals. Overall consumers are concerned that animal cloning is an unnatural process and that it will lead to human cloning. Copyright © 2011 Elsevier Ltd. All rights reserved.

Knowledge and attitudes toward human cloning in Israel.

PubMed

Barnoy, Sivia; Ehrenfeld, Malka; Sharon, Rina; Tabak, Nili

2006-04-01

The success of mammal cloning in 1997 has brought the issue of human cloning into public discussion. Human cloning has several aspects and potential applications for use in both reproductive and non-reproductive matters. The aim of this study was to evaluate the knowledge and attitudes toward human cloning in Israel. Data from 120 respondents (68 health professionals and 52 non-health professionals), all Jewish, Hebrew speaking with at least 15 years of education each, were collected using two questionnaires that dealt with knowledge and attitudes toward human cloning. Results showed that although health professionals had significantly more knowledge that non-health professionals, all respondents had poor knowledge about cloning. No difference in attitudes was found between the groups. Most respondents opposed human cloning, but more positive attitudes toward non-reproductive cloning were found. The results are discussed in the context of the deficit model. The findings indicate a need to provide information about human cloning to allow people to form their attitudes based on factual knowledge.

Influence of spatial distribution and size of clones on the realized outcrossing rate of the marsh cinquefoil (Comarum palustre)

PubMed Central

Somme, L.; Mayer, C.; Raspé, O.; Jacquemart, A.-L.

2014-01-01

Background and Aims Clonal growth is a common feature in flowering plants. As clone size increases, the selfing rate in self-compatible species is likely to increase due to more frequent geitono-pollination events (i.e. pollination among flowers within the same genet). This study investigated the breeding system of the marsh cinquefoil (Comarum palustre) and assessed spatial distribution of clones, clone size and architecture, and their effects on realized outcrossing rates. In addition, pollen dispersal was investigated in two patchy populations. Methods The species' breeding system was investigated under controlled conditions through hand pollinations (self- vs. cross-pollination). Using microsatellite markers, an assessment was made of the realized outcrossing rates and the genetic diversity in four natural populations, the clonal structure in two populations within five 15 × 15 m sampling plots following 0·5 × 0·5 m grids, and the pollen dispersal through paternity assignment tests in those two populations. Key Results Comarum palustre is a self-compatible species but only presents a low rate of spontaneous self-pollination. The occurrence of inbreeding depression was not detected at the seed set stage (δSS = 0·04). Clones were spatially clumped (AC = 0·60–0·80), with intermediate to no intermingling of the ramets (DC = 0·40–1·00). Genet size ranged from one to 171 ramets. Patchy populations had low outcrossing rates (tm = 0·33–0·46). Large clones showed lower outcrossing rates than small clones. Pollen dispersal mainly occurred within patches as only 1–7 % of the pollination events occurred between patches of >25 m separation. Seedling recruitment events were detected. Conclusions Genet size together with distances between patches, through increasing geitono-pollination events, appeared to be important factors influencing realized outcrossing rates. The study also revealed seed flow allowing seedling recruitment, which may contribute to increasing the number of new patches, and potentially further enhance gene flow within populations. PMID:24284813

Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines.

PubMed

Agheli, Reza; Emkanian, Bijan; Halabian, Raheleh; Fallah Mehrabadi, Jalil; Imani Fooladi, Abbas Ali

2017-02-01

About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/µL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/µL and 1000 ng/µL ( P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon γ and tumor necrosis factor α compared to control. According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.

Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

PubMed

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H

2013-06-01

The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

Cloning Changes the Response to Obesity of Innate Immune Factors in Blood, Liver, and Adipose Tissues in Domestic Pigs

PubMed Central

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan

2013-01-01

Abstract The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity. PMID:23668862

Ethical attitudes on human cloning among professionals in Taiwan and the policy implications for regulation.

PubMed

Yang, Che-Ming; Chung, Chun-Chih; Lu, Meei-Shiow; Lin, Chiou-Fen; Chen, Jiun-Shyan

2005-01-01

This research focused on understanding the attitudes toward human cloning in Taiwan among professionals in healthcare, law, and religion. The study was conducted utilizing a structured questionnaire. 220 healthcare professionals from two regional hospitals located in Taipei, 351 religious professionals in the northern Taiwan and 711 legal professionals were selected by to receive questionnaires. The valid response rate is 42.1% The questions were generated by an expert panel and represented major arguments in the human cloning debate. There were a total of six Likert scaled questions in the questionnaire. The responses were coded from 1 to 5 with 1 representing strong opposition to human cloning, 3 representing a neutral attitude; and 5 representing a strong favorable attitude toward human cloning. Healthcare professionals had the highest overall average score of 2.14 and the religious professionals had the lowest average at 1.58. All three categories of respondents' attitude toward cloning ranged from mild opposition to strong opposition to human cloning. The religious professionals were more strongly opposed to cloning. Age, education, and religion significantly influenced attitudes toward cloning. Professionals between fifty-one and sixty years old, those with less education, and Roman Catholic professionals were more strongly opposed to cloning. Religious professionals were more strongly opposed to human cloning than professionals in healthcare or law. Younger professionals as an age group demonstrated less opposition to human cloning. Regulation of human cloning will be influenced by professionals in healthcare, law, and religion, and the regulatory environment chosen now will play a pivotal role in influencing the acceptance of human cloning in the future.