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Sample records for recombination dna vector

  1. Direct facile screening of recombinant DNA vector constructs.

    PubMed

    Winnard, Paul T; Challa, Rushi; Bhujwalla, Zaver M; Raman, Venu

    2014-04-01

    Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.

  2. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

    PubMed Central

    Kitts, P A; Ayres, M D; Possee, R D

    1990-01-01

    Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location. Images PMID:2216760

  3. Recombinant DNA cloning vectors containing selectable genetic markers for use in streptomyces and related organisms

    SciTech Connect

    Mabe, J.A.; Nakatsukasa, W.M.

    1987-10-06

    This patent describes a recombinant DNA cloning vector comprising: (a) a functional origin of replication-containing restriction fragment of plasmid pMND1000, and (b) one or more DNA segments that convey resistance to at least one antibiotic or that convey colorimetric sensitivity when transformed into a sensitive restrictionless host cell.

  4. Recombinant DNA cloning vectors containing selectable genetic markers for use in streptomyces and related organisms

    SciTech Connect

    Mabe, J.A.; Nakasukasa, W.M.

    1987-07-28

    A recombinant DNA cloning vector is described comprising: (a) a functional origin of replication-containing restriction fragment of plasmid pMND900, and (b) one or more DNA segments that convey resistance to at least one antibiotic or that convey colorimetric sensitivity when transformed into a sensitive restrictionless host cell.

  5. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  6. Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.

    PubMed Central

    Duboise, S M; Guo, J; Desrosiers, R C; Jung, J U

    1996-01-01

    We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells. Images Fig. 3 PMID:8876145

  7. A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.

    PubMed

    Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andréasson, Claes

    2014-06-01

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs.

  8. Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion.

    PubMed Central

    Olson, P; Temin, H M; Dornburg, R

    1992-01-01

    Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences. Images PMID:1310753

  9. A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning.

    PubMed

    Ayabe, Fumiaki; Katoh, Motonobu; Inoue, Toshiaki; Kouprina, Natalay; Larionov, Vladimir; Oshimura, Mitsuo

    2005-01-01

    Following the recent completion of the human genome sequence, genomics research has shifted its focus to understanding gene complexity, expression, and regulation. However, in order to investigate such issues, there is a need to develop a practical system for genomic DNA expression. Transformation-associated recombination (TAR) cloning has proven to be a convenient tool for selective isolation of a genetic locus from a complex genome as a circular YAC using recombination in yeast. The human artificial chromosome (HAC) vector containing an acceptor loxP site has served as a platform for the reproducible expression of transgenes. In this study, we describe a system that efficiently expresses a genetic locus in mammalian cells by retrofitting a TAR-YAC with the donor loxP site and loading it onto the HAC vector by the Cre/loxP system. In order to demonstrate functional expression of genomic loci, the entire human hypoxanthine phosphoribosyl transferase (HPRT) locus contained in a 100 kb YAC was loaded onto the HAC vector and was shown to complement the genetic defect in Hprt-deficient CHO cells. Thus, the combination of TAR cloning and the HAC vector may serve as a powerful tool for functional genomic studies.

  10. Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination.

    PubMed

    Minke, J M; Fischer, L; Baudu, Ph; Guigal, P M; Sindle, T; Mumford, J A; Audonnet, J C

    2006-05-15

    In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.

  11. Construction of gene-targeting vectors by recombineering.

    PubMed

    Lee, Song-Choon; Wang, Wei; Liu, Pentao

    2009-01-01

    Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by gamma phage to manipulate DNA in Escherichia coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.

  12. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    PubMed

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

  13. Integration-free reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) without viral vectors, recombinant DNA, and genetic modification.

    PubMed

    Heng, Boon Chin; Fussenegger, Martin

    2014-01-01

    Stem cells are envisaged to be integral components of multicellular systems engineered for therapeutic applications. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) via recombinant expression of a limited number of transcription factors, which was first achieved by Yamanaka and colleagues in 2007, heralded a major breakthrough in the stem cell field. Since then, there has been rapid progress in the field of iPSC generation, including the identification of various small molecules that can enhance reprogramming efficiency and reduce the number of different transcription factors required for reprogramming. Nevertheless, the major obstacles facing clinical applications of iPSCs are safety concerns associated with the use of viral vectors and recombinant DNA for expressing the appropriate transcription factors during reprogramming. In particular, permanent genetic modifications to newly reprogrammed iPSCs have to be avoided in order to meet stringent safety requirements for clinical therapy. These safety challenges can be overcome by new technology platforms that enable cellular reprogramming to iPSCs without the need to utilize either recombinant DNA or viral vectors. The use of recombinant cell-penetrating peptides and direct transfection of synthetic mRNA encoding appropriate transcription factors have both been shown to successfully reprogram somatic cells to iPSCs. It has also been shown more recently that the direct transfection of certain miRNA species can reprogram somatic cells to pluripotency without the need for any of the transcription factors commonly utilized for iPSC generation. This chapter describes protocols for iPSC generation with these new techniques, which would obviate the use of recombinant DNA and viral vectors in cellular reprogramming, thus avoiding permanent genetic modification to the reprogrammed cells.

  14. Nonreplicating vaccinia vector efficiently expresses recombinant genes.

    PubMed

    Sutter, G; Moss, B

    1992-11-15

    Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

  15. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  16. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  17. Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

    PubMed Central

    2011-01-01

    Background There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. Results This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody

  18. Analysis of Partial Recombinants in Lentiviral Vector Preparations

    PubMed Central

    Kuate, Seraphin; Marino, Michael P.

    2014-01-01

    Abstract The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 106 transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished. PMID:24367910

  19. NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee.

    PubMed

    O'Reilly, Marina; Shipp, Allan; Rosenthal, Eugene; Jambou, Robert; Shih, Tom; Montgomery, Maureen; Gargiulo, Linda; Patterson, Amy; Corrigan-Curay, Jacqueline

    2012-01-01

    In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.

  20. A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.

    PubMed

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2012-12-01

    Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure.

  1. A Pilot Study Comparing the Development of EIAV Env-Specific Antibodies Induced by DNA/Recombinant Vaccinia-Vectored Vaccines and an Attenuated Chinese EIAV Vaccine

    PubMed Central

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian

    2012-01-01

    Abstract Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAVFDDV. Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure. PMID:23171359

  2. Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

    PubMed Central

    Aoki, K.; Barker, C.; Danthinne, X.; Imperiale, M. J.; Nabel, G. J.

    1999-01-01

    BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research. Images Fig. 1 Fig. 2 Fig. 4 PMID:10448644

  3. Construction and expression of recombined human AFP eukaryotic expression vector

    PubMed Central

    Zhang, Li-Wang; Ren, Jun; Zhang, Liang; Zhang, Hong-Mei; Jin, Bin; Pan, Bo-Rong; Si, Xiao-Ming; Zhang, Yan-Jun; Wang, Zhong-Hua; Pan, Yang-Lin; Festein, Stephen M

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/mL AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P < 0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma. PMID:12854142

  4. Recombinant Salmonella Bacteria Vectoring HIV/AIDS Vaccines.

    PubMed

    Chin'ombe, Nyasha; Ruhanya, Vurayai

    2013-01-01

    HIV/AIDS is an important public health problem globally. An affordable, easy-to-deliver and protective HIV vaccine is therefore required to curb the pandemic from spreading further. Recombinant Salmonella bacteria can be harnessed to vector HIV antigens or DNA vaccines to the immune system for induction of specific protective immunity. These are capable of activating the innate, humoral and cellular immune responses at both mucosal and systemic compartments. Several studies have already demonstrated the utility of live recombinant Salmonella in delivering expressed foreign antigens as well as DNA vaccines to the host immune system. This review gives an overview of the studies in which recombinant Salmonella bacteria were used to vector HIV/AIDS antigens and DNA vaccines. Most of the recombinant Salmonella-based HIV/AIDS vaccines developed so far have only been tested in animals (mainly mice) and are yet to reach human trials.

  5. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis.

    PubMed

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

    2015-11-27

    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development.

  6. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis

    PubMed Central

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

    2015-01-01

    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development. PMID:26403370

  7. Priming Immunization with DNA Augments Immunogenicity of Recombinant Adenoviral Vectors for Both HIV-1 Specific Antibody and T-Cell Responses

    PubMed Central

    Koup, Richard A.; Roederer, Mario; Lamoreaux, Laurie; Fischer, Jennifer; Novik, Laura; Nason, Martha C.; Larkin, Brenda D.; Enama, Mary E.; Ledgerwood, Julie E.; Bailer, Robert T.; Mascola, John R.; Nabel, Gary J.; Graham, Barney S.

    2010-01-01

    Background Induction of HIV-1-specific T-cell responses relevant to diverse subtypes is a major goal of HIV vaccine development. Prime-boost regimens using heterologous gene-based vaccine vectors have induced potent, polyfunctional T cell responses in preclinical studies. Methods The first opportunity to evaluate the immunogenicity of DNA priming followed by recombinant adenovirus serotype 5 (rAd5) boosting was as open-label rollover trials in subjects who had been enrolled in prior studies of HIV-1 specific DNA vaccines. All subjects underwent apheresis before and after rAd5 boosting to characterize in depth the T cell and antibody response induced by the heterologous DNA/rAd5 prime-boost combination. Results rAd5 boosting was well-tolerated with no serious adverse events. Compared to DNA or rAd5 vaccine alone, sequential DNA/rAd5 administration induced 7-fold higher magnitude Env-biased HIV-1-specific CD8+ T-cell responses and 100-fold greater antibody titers measured by ELISA. There was no significant neutralizing antibody activity against primary isolates. Vaccine-elicited CD4+ and CD8+ T-cells expressed multiple functions and were predominantly long-term (CD127+) central or effector memory T cells and that persisted in blood for >6 months. Epitopes mapped in Gag and Env demonstrated partial cross-clade recognition. Conclusion Heterologous prime-boost using vector-based gene delivery of vaccine antigens is a potent immunization strategy for inducing both antibody and T-cell responses. Trial Registration ClinicalTrails.gov NCT00102089, NCT00108654 PMID:20126394

  8. Mucosal immunotherapy in an Alzheimer mouse model by recombinant Sendai virus vector carrying Aβ1-43/IL-10 cDNA.

    PubMed

    Hara, Hideo; Mouri, Akihiro; Yonemitsu, Yoshikazu; Nabeshima, Toshitaka; Tabira, Takeshi

    2011-10-06

    Based on the amyloid cascade hypothesis, many reports have indicated that immunotherapy is beneficial for Alzheimer's disease (AD). We developed a mucosal immunotherapy for AD by nasal administration of recombinant Sendai virus vector carrying Aβ1-43 and mouse IL-10 cDNA. Nasal but not intramuscular administration of the vaccine induced good antibody responses to Aβ. When APP transgenic mice (Tg2576) received this vaccine once nasally, the Aβ plaque burden was significantly decreased 8 weeks after without inducing inflammation in the brain. The amount of Aβ measured by ELISA was also reduced in both soluble and insoluble fractions of the brain homogenates, and notably the Aβ oligomer (12-mer) was also apparently decreased. Tg2576 mice showed significant improvement in cognitive functions examined at 3 months after vaccination. Thus, this is an alternative immunotherapy for AD, which has an advantage in non-invasive, safe and relatively long lasting features.

  9. Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression.

    PubMed

    Krupa, Magdalena; Canamero, Marta; Gomez, Carmen E; Najera, Jose L; Gil, Jesus; Esteban, Mariano

    2011-02-04

    Despite recent advances in early detection and improvement of conventional therapies, there is an urgent need for development of additional approaches for prevention and/or treatment of prostate cancer, and the use of immunotherapeutic modalities, such as cancer vaccines, is one of the most promising strategies. In this study, we evaluated the prophylactic efficacy of an active immunization protocol against prostate cancer associated antigens mPSCA and mSTEAP1 in experimental prostate cancer. Two antigen delivery platforms, recombinant DNA and MVA vectors, both encoding either mPSCA or mSTEAP1 were used in diversified DNA prime/MVA boost vaccination protocol. Antitumour activity was evaluated in TRAMP-C1 subcutaneous syngeneic tumour model and TRAMP mice. DNA prime/MVA boost immunization against either mPSCA or mSTEAP1, delayed tumour growth in TRAMP-C1 cells-challenged mice. Furthermore, simultaneous vaccination with both antigens produced a stronger anti-tumour effect against TRAMP-C1 tumours than vaccination with either mPSCA or mSTEAP1 alone. Most importantly, concurrent DNA prime/MVA boost vaccination regimen with those antigens significantly decreased primary tumour burden in TRAMP mice without producing any apparent adverse effects. Histopathological analysis of prostate tumours from vaccinated and control TRAMP mice revealed also that mPSCA/mSTEAP1 based-vaccination was effective at reducing the severity of prostatic lesions and incidence of high-grade poorly differentiated prostate cancer. Suppression of the disease progression in TRAMP mice was correlated with decreased proliferation index and increased infiltration of T-cells in prostate tissue. Active immunization against PSCA and STEAP1 using DNA prime/MVA boost strategy is a promising approach for prevention and/or treatment of prostate cancer.

  10. Herpes simplex virus type 1-derived recombinant and amplicon vectors.

    PubMed

    Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

  11. Construction of human artificial chromosome vectors by recombineering.

    PubMed

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

    2005-05-23

    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  12. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  13. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  14. Recombinant DNA products: Insulin, interferon and growth hormone

    SciTech Connect

    Bollon, A.P.

    1984-01-01

    This book provides the discussion of products of biotechnology of recombinant DNA. The contents include: Recombinant DNA techniques; isolation, cloning, and expression of genes; from somatostatin to human insulin; yeast; an alternative organism for foreign protein production; background in human interferon; preclinical assessment of biological properties of recombinant DNA derived human interferons; human clinical trials of bacteria-derived human ..cap alpha.. interferon.f large scale production of human alpha interferon from bacteria; direct expression of human growth hormone in escherichia coli with the lipoprotein promoter; biological actions in humans of recombinant DNA synthesized human growth hormone; NIH guidelines for research involving recombinant DNA molecules; appendix; viral vectors and the NHY guidelines; FDA's role in approval and regulation of recombinant DNA drugs; and index.

  15. HSV Recombinant Vectors for Gene Therapy

    PubMed Central

    Manservigi, Roberto; Argnani, Rafaela; Marconi, Peggy

    2010-01-01

    The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges. PMID:20835362

  16. Recombinant DNA. Part F

    SciTech Connect

    Wu, R.

    1987-01-01

    This book contains 36 papers. Some of the titles are: Site-specific cleavage of DNA at 8-, 9-, and 10-bp sequences; The use of DNA methylases to alter the apparent recognition specificities of restriction endonucleases; Random cloning and sequencing by the M13/Dideoxynucleotide chain termination method; Solid-phase methods for sequencing of oligodeoxynucleotides and DNA; and Analysis of modified nucleosides and nucleotide sequence of tRNA.

  17. Sustained expression from DNA vectors.

    PubMed

    Wong, Suet Ping; Argyros, Orestis; Harbottle, Richard P

    2015-01-01

    DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

  18. Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector.

    PubMed

    Lucifora, Julie; Salvetti, Anna; Marniquet, Xavier; Mailly, Laurent; Testoni, Barbara; Fusil, Floriane; Inchauspé, Aurore; Michelet, Maud; Michel, Marie-Louise; Levrero, Massimo; Cortez, Pierre; Baumert, Thomas F; Cosset, François-Loic; Challier, Cécile; Zoulim, Fabien; Durantel, David

    2017-09-01

    Hepatitis B Virus (HBV) persists in infected hepatocytes as an episomal covalently-closed-circular DNA mini-chromosome, called cccDNA. As the main nuclear transcription template, HBV cccDNA is a key replication intermediate in the viral life cycle. Little is known about the mechanisms involved in its formation, maintenance and fate under antiviral therapies. This is mainly due to the lack of small immune-competent animal models able to recapitulate the entire HBV replication cycle, including formation of HBV cccDNA. Here we report that HBV cccDNA can be detected by Southern blot analyses in the liver of C57BL6 mice transduced with AAV-HBV. HBV cccDNA persists in the liver of these animals together with the AAV-HBV episome. We also set up a PCR strategy to distinguish the HBV cccDNA from the AAV-HBV episome. These suggest that the AAV-HBV/mouse model might be relevant to test drugs targeting HBV cccDNA regulation and persistence. Copyright © 2017. Published by Elsevier B.V.

  19. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  20. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) ACTION: Notice of consideration of proposed...- vector system may be certified only after review by the NIH Recombinant DNA Advisory Committee (RAC) and...

  1. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  2. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  3. Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

    PubMed

    Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development.

  4. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    PubMed

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.

  5. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  6. Biosafety of recombinant adeno-associated virus vectors.

    PubMed

    Dismuke, David J; Tenenbaum, Liliane; Samulski, R Jude

    2013-12-01

    It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

  7. Recombinant DNA technology in apple.

    PubMed

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available.

  8. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  9. One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

    PubMed

    Li, Li-Li; Wang, He-Rong; Zhou, Zhi-Yi; Luo, Jing; Xiao, Xiang-Qian; Wang, Xiao-Li; Li, Jin-Tao; Zhou, Yu-Bai; Zeng, Yi

    2016-04-01

    Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections.

  10. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  11. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  12. A novel DNA selection and direct extraction process and its application in DNA recombination.

    PubMed

    Wang, An-Bang; Cheng, Chia-Wei; Lin, I-Chun; Lu, Fei-Yau; Tsai, Huai-Jen; Lin, Chiu-Chun; Yang, Chun-Hui; Pan, Po-Ting; Kuan, Chen-Chi; Chen, Yen-Chih; Lin, Yi-Wei; Chang, Chih-Ning; Wu, Yi-Hung; Kurniawan, Tetuko; Lin, Chii-Wann; Wo, Andrew M; Chen, Lin-Chi

    2011-02-01

    In the conventional bench-top approach, the DNA recombination process is time- and effort-consuming due to laborious procedures lasting from several hours to a day. A novel DNA selection and direct extraction process has been proposed, integrated and tested on chip. The integrative microfluidic chip can perform the whole procedure of DNA recombination, including DNA digestion, gel electrophoresis, DNA extraction and insert-vector ligation within 1 h. In this high-throughput design, the manual gel cutting was replaced by an automatic processing system that performed high-quality and high-recovery efficiency in DNA extraction process. With no need of gel-dissolving reagents and manipulation, the application of selection and direct extraction process could significantly eliminate the risks from UV and EtBr and also facilitate DNA recombination. Reliable output with high success rate of cloning has been achieved with a significant reduction in operational hazards, required materials, efforts and time.

  13. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

  14. 8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

    PubMed Central

    Meira, L B; Henriques, J A; Magaña-Schwencke, N

    1995-01-01

    The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Images PMID:7784218

  15. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  16. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

    PubMed

    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate.

  17. Strategies for RNA-Guided DNA Recombination

    NASA Astrophysics Data System (ADS)

    Angeleska, Angela; Jonoska, Nataša; Saito, Masahico; Landweber, Laura F.

    We present a model for homologous DNA recombination events guided by double-stranded RNA (dsRNA) templates, and apply this model to DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha. In these organisms, differentiation of a somatic macronucleus from a germline micronucleus involves extensive gene rearrangement, which can be modeled as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a graph structure, which we refer to as an assembly graph, containing only 1- and 4-valent vertices can provide a physical representation of the DNA at the time of recombination. With this representation, 4-valent vertices correspond to the alignment of the recombination sites, and we model the actual recombination event as smoothing of these vertices.

  18. Disease diagnosis by recombinant DNA methods

    SciTech Connect

    Caskey, C.T.

    1987-06-05

    Recombinant DNA procedures have now been applied to the problem of the identification of molecular defects in man that account for heritable diseases, somatic mutations associated with neoplasia, and acquired infectious disease. Thus, recombinant DNA technology has rapidly expanded the ability to diagnose disease. Substantial advances in the simplification of procedures for diagnostic purposes have been made, and the informed physician has gained in diagnostic accuracy as a consequence of these developments. The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment. 66 references, 7 figures.

  19. [Recombinant viruses of poultry as vector vaccines against fowl plague].

    PubMed

    Fuchs, Walter; Veits, Jutta; Mettenleiter, Thomas C

    2006-01-01

    To help in the control of fowl plague caused by highly pathogenic avian influenza A viruses of hemagglutinin (HA) subtypes H5 and H7 several vaccines have been developed. A prophylactic immunization of poultry with inactivated influenza viruses in non-endemic situations is questionable, however, due to the impairment of serological identification of field virus-infected animals which hinders elimination of the infectious agent from the population. This problem might be overcome by the use of genetically engineered marker vaccines which contain only the protective influenza virus hemagglutinin. Infected animals could then be unambiguously identified by their serum antibodies against other influenza virus proteins, e.g. neuraminidase or nucleoprotein. For such a use, purified HA or HA-expressing DNA vaccines are conceivable. Economically advantageous and easier to apply are modified live virus vaccines in use against other poultry diseases, which have been modified to express influenza virus HA. So far, recombinant HA-expressing fowlpox virus (FPV) as well as infectious laryngotracheitis and Newcastle disease viruses have been asssessed in animal experiments. An H5-expressing FPV recombinant is already in use in Central America and Southeast Asia but without accompanying marker diagnostics. Advantages and disadvantages of the different viral vectors are discussed.

  20. Construction, production, and purification of recombinant adenovirus vectors.

    PubMed

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel

    2014-01-01

    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  1. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  2. Recombination in eukaryotic single stranded DNA viruses.

    PubMed

    Martin, Darren P; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-09-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  3. An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA

    PubMed Central

    Deuring, Renate; Klotz, Günther; Doerfler, Walter

    1981-01-01

    variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis. Images PMID:6265937

  4. Alphavirus vectors: applications for DNA vaccine production and gene expression.

    PubMed

    Lundstrom, K

    2000-01-01

    Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

  5. Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

    PubMed

    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

    2015-01-08

    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

  6. DNA recombination. Recombination initiation maps of individual human genomes.

    PubMed

    Pratto, Florencia; Brick, Kevin; Khil, Pavel; Smagulova, Fatima; Petukhova, Galina V; Camerini-Otero, R Daniel

    2014-11-14

    DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

  7. Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR)

    PubMed Central

    Newman, Robert J.; Roose-Girma, Merone; Warming, Søren

    2015-01-01

    A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described. We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette. The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step. PMID:26089387

  8. A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo.

    PubMed

    Lu, Jiamiao; Williams, James A; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A

    2017-01-01

    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.

  9. Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

    PubMed

    Osada, Takuya; Morse, Michael A; Hobeika, Amy; Lyerly, H Kim

    2012-06-01

    Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed.

  10. Design and generation of recombinant rabies virus vectors

    PubMed Central

    Osakada, Fumitaka; Callaway, Edward M.

    2014-01-01

    Rabies viruses, negative-strand RNA viruses, infect neurons through axon terminals and spread transsynaptically in a retrograde direction between neurons. Rabies viruses whose glycoprotein (G) gene is deleted from the genome cannot spread across synapses. Complementation of G in trans, however, enables transsynaptic spreading of G-deleted rabies viruses to directly-connected, presynaptic neurons. Recombinant rabies viruses can encode genes of interest for labeling cells, controlling gene expression, and monitoring or manipulating neural activity. Cre-dependent or bridge-protein-mediated transduction and single-cell electroporation via EnvA/TVA or EnvB/TVB system allow cell-type-specific or single-cell-specific targeting. These rabies virus-based approaches permit the linking of connectivity to cell morphology and circuit function for particular cell types or single cells. Here we describe methods for construction of rabies viral vectors, recovery of G-deleted rabies viruses from cDNA, amplification of the viruses, pseudotyping them with EnvA or EnvB, and concentration and titration of the viruses. The entire protocol takes 6–8 weeks. PMID:23887178

  11. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

    PubMed

    Lu, Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  12. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    PubMed

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  13. DNA Sequence Alignment during Homologous Recombination*

    PubMed Central

    Greene, Eric C.

    2016-01-01

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  14. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. [Arteriogenesis induced by intramyocardial recombinant adeno-associated virus vector encoding human CD151 cDNA gene transfer in swines with coronary artery occlusion].

    PubMed

    Zuo, Hou-juan; Liu, Zheng-xiang; Liu, Xiao-chun; Zeng, He-song; Wen, Sha; Liu, Tao; Wang, Dao-wen; Zhang, Xin

    2009-06-01

    To investigate the efficacy of CD151 gene delivery in promoting blood perfusion in swines after myocardial infarction. Swines received coronary artery ligation and intramyocardial injection with rAAV-CD151, rAAV-anti-CD151 or rAAV-GFP. Eight weeks after vector injection, Western blot, immunostaining and 13N-labeled NH3 PET were performed to detect gene expression and biological effects of various treatments. High level of CD151 protein expression was detected in the rAAV-CD151 group. The capillary density in the rAAV-CD151 group [(83.8 +/- 6.7) n/mm2] was significantly higher than that in the control group [(33.2 +/- 4.5) n/mm2] and rAAV-GFP group [(41.6 +/- 5.6) n/mm2] (all P<0.05); the arteriole density in the rAAV-CD151 group [(16.4 +/- 2.5) n/mm2] was also higher than that in the control group [(6.6 +/- 2.3) n/mm2] and the rAAV-GFP group [(8.4 +/- 1.6) n/mm2] (all P<0.05). However, the lowest capillary density and arteriole density were evidenced in rAAV-anti-CD151 group. Myocardial blood perfusion was significantly increased in rAAV-CD151 group and significantly reduced in rAAV-anti-CD151 group (all P<0.05 vs. control). Intramyocardial injection of rAAV-CD151 could enhance the myocardial express of CD151 protein, increase capillary and arteriole densities and improve blood perfusion in swine with myocardial infarction.

  16. Adenoviral vector DNA for accurate genome editing with engineered nucleases.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Henriques, Sara F D; Janssen, Josephine M; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-10-01

    Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

  17. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  18. Transcription and recombination: when RNA meets DNA.

    PubMed

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Science: The Recombinant DNA Advisory Committee.

    ERIC Educational Resources Information Center

    Wright, Susan

    1979-01-01

    Reports on the status of the Recombinant DNA Advisory Committee (RAC) and attempts to rationalize Suburban Highway Policy. Effective communication among members of the RAC is a current problem facing the committee. A federal transportation priority spending policy is suggested during these times of money and fuel shortages. (MA)

  20. Recombinant DNA: Scientific and Social Perspectives.

    ERIC Educational Resources Information Center

    Vandegrift, Vaughn

    1979-01-01

    This article is designed to inform chemical educators not engaged in this technology as to the nature and methods used in the technology, the reasons for scientific and social concern, and the attempts made to assuage concerns involving recombinant DNA research. (author/BB)

  1. Recombinant DNA: Scientific and Social Perspectives.

    ERIC Educational Resources Information Center

    Vandegrift, Vaughn

    1979-01-01

    This article is designed to inform chemical educators not engaged in this technology as to the nature and methods used in the technology, the reasons for scientific and social concern, and the attempts made to assuage concerns involving recombinant DNA research. (author/BB)

  2. Vaccine development using recombinant DNA technology

    USDA-ARS?s Scientific Manuscript database

    Vaccines induce an immune response in the host that subsequently recognizes infectious agents and helps fight off the disease; vaccines must do this without causing the disease. This paper reviews the development of recombinant DNA technologies as a means of providing new ways for attenuating diseas...

  3. Science: The Recombinant DNA Advisory Committee.

    ERIC Educational Resources Information Center

    Wright, Susan

    1979-01-01

    Reports on the status of the Recombinant DNA Advisory Committee (RAC) and attempts to rationalize Suburban Highway Policy. Effective communication among members of the RAC is a current problem facing the committee. A federal transportation priority spending policy is suggested during these times of money and fuel shortages. (MA)

  4. Recombination spots prediction using DNA physical properties in the saccharomyces cerevisiae genome

    NASA Astrophysics Data System (ADS)

    Guo, Shou-Hui; Xu, Li-Qin; Chen, Wei; Liu, Guo-Qing; Lin, Hao

    2012-09-01

    The prediction of meiotic recombination is difficult and current available methods are limited. In this study, we propose a novel method for discriminating between recombination hotspots and coldspots using support vector machine(SVM) with the DNA physical properties. Results of optimized pseudo-tetranucleotide show overall accuracy of 83.1% by using 5-fold cross-validation. High predictive successful rate exhibit that this model can be applied for discriminating between recombination hotspots and coldspots.

  5. Distribution of Brugia malayi larvae and DNA in vector and non-vector mosquitoes: implications for molecular diagnostics

    PubMed Central

    2009-01-01

    Background The purpose of this study was to extend prior studies of molecular detection of Brugia malayi DNA in vector (Aedes aegypti- Liverpool) and non-vector (Culex pipiens) mosquitoes at different times after ingestion of infected blood. Results Parasite DNA was detected over a two week time course in 96% of pooled thoraces of vector mosquitoes. In contrast, parasite DNA was detected in only 24% of thorax pools from non-vectors; parasite DNA was detected in 56% of midgut pools and 47% of abdomen pools from non-vectors. Parasite DNA was detected in vectors in the head immediately after the blood meal and after 14 days. Parasite DNA was also detected in feces and excreta of the vector and non-vector mosquitoes which could potentially confound results obtained with field samples. However, co-housing experiments failed to demonstrate transfer of parasite DNA from infected to non-infected mosquitoes. Parasites were also visualized in mosquito tissues by immunohistololgy using an antibody to the recombinant filarial antigen Bm14. Parasite larvae were detected consistently after mf ingestion in Ae. aegypti- Liverpool. Infectious L3s were seen in the head, thorax and abdomen of vector mosquitoes 14 days after Mf ingestion. In contrast, parasites were only detected by histology shortly after the blood meal in Cx. pipiens, and these were not labeled by the antibody. Conclusion This study provides new information on the distribution of filarial parasites and parasite DNA in vector and non-vector mosquitoes. This information should be useful for those involved in designing and interpreting molecular xenomonitoring studies. PMID:19922607

  6. Characterization of recombinant Raccoonpox Vaccine Vectors in Chickens

    USGS Publications Warehouse

    Hwa, S.-H.; Iams, Keith P.; Hall, Jeffrey S.; Kingstad, B.A.; Osorio, Jorge E.

    2010-01-01

    Raccoonpox virus (RCN) has been used as a recombinant vector against several mammalian pathogens but has not been tested in birds. The replication of RCN in chick embryo fibroblasts (CEFs) and chickens was studied with the use of highly pathogenic avian influenza virus H5N1 hemagglutinin (HA) as a model antigen and luciferase (luc) as a reporter gene. Although RCN replicated to low levels in CEFs, it efficiently expressed recombinant proteins and, in vivo, elicited anti-HA immunoglobulin yolk (IgY) antibody responses comparable to inactivated influenza virus. Biophotonic in vivo imaging of 1-wk-old chicks with RCN-luc showed strong expression of the luc reporter gene lasting up to 3 days postinfection. These studies demonstrate the potential of RCN as a vaccine vector for avian influenza and other poultry pathogens. ?? American Association of Avian Pathologists 2010.

  7. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo.

    PubMed

    Zhong, L; Li, W; Yang, Z; Chen, L; Li, Y; Qing, K; Weigel-Kelley, K A; Yoder, M C; Shou, W; Srivastava, A

    2004-07-01

    Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.

  8. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

    PubMed Central

    Simon, J R; Moore, P D

    1987-01-01

    Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. Images PMID:3302673

  9. Monitoring DNA recombination initiated by HO endonuclease.

    PubMed

    Sugawara, Neal; Haber, James E

    2012-01-01

    DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.

  10. Recombinant adeno-associated viral vector reference standards.

    PubMed

    Moullier, Philippe; Snyder, Richard O

    2012-01-01

    Reference standard materials (RSMs) exist for a variety of biologics including vaccines but are not readily available for gene therapy vectors. To date, a recombinant adeno-associated virus serotype 2 RSM (rAAV2 RSM) has been produced and characterized and was made available to the scientific community in 2010. In addition, a rAAV8 RSM has been produced and will be characterized in the coming months. The use of these reference materials by members of the gene therapy field facilitates the calibration of individual laboratory vector-specific internal standards and the eventual comparison of preclinical and clinical data based on common dosage units. Normalization of data to determine therapeutic dose ranges of rAAV vectors for each particular tissue target and disease indication is important information that can enhance the safety and protection of patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination.

    PubMed Central

    Tsuzuki, T; Rancourt, D E

    1998-01-01

    Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information. PMID:9461458

  12. Recombination-dependent concatemeric viral DNA replication.

    PubMed

    Lo Piano, Ambra; Martínez-Jiménez, María I; Zecchi, Lisa; Ayora, Silvia

    2011-09-01

    The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.

  13. Clinical gene therapy using recombinant adeno-associated virus vectors.

    PubMed

    Mueller, C; Flotte, T R

    2008-06-01

    Recombinant adeno-associated virus (rAAV) vectors possess a number of properties that may make them suitable for clinical gene therapy, including being based upon a virus for which there is no known pathology and a natural propensity to persist in human cells. Wild-type adeno-associated viruses (AAVs) are now known to be very diverse and ubiquitous in humans and nonhuman primates, which adds to the degree of confidence one may place in the natural history of AAV, namely that it has never been associated with any human tumors or other acute pathology, other than sporadic reports of having been isolated from spontaneously aborted fetuses. On the basis of this understanding of AAV biology and a wide range of preclinical studies in mice, rabbits, dogs and nonhuman primates, a growing number of clinical trials have been undertaken with this class of vectors. Altogether, over 40 clinical trials have now been approved. Although all previous trials were undertaken using AAV serotype 2 vectors, at least two current trials utilize AAV2 vector genomes cross-packaged or pseudotyped into AAV1 capsids, which appear to mediate more efficient gene delivery to muscle. The explosion of capsid isolates available for use as vectors to over 120 has now provided the potential to broaden the application of AAV-based gene therapy to other cell types.

  14. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    PubMed

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  15. A recombineering based approach for high-throughput conditional knockout targeting vector construction

    PubMed Central

    Chan, Waiin; Costantino, Nina; Li, Ruixue; Lee, Song Choon; Su, Qin; Melvin, David; Court, Donald L.; Liu, Pentao

    2007-01-01

    Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies. PMID:17426124

  16. EASE vectors for rapid stable expression of recombinant antibodies.

    PubMed

    Aldrich, Teri L; Viaje, Aurora; Morris, Arvia E

    2003-01-01

    Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models. Typically, this amount of recombinant protein expression involves large numbers of transient transfections or cloning of a recombinant cell line. Both of these approaches are time-consuming and labor-intensive. In this report, we describe the application of an EASE vector system that is capable of generating stable pools of transfected Chinese hamster ovary cells. These pooled populations of cells produce high quantities of antibody candidates without labor-intensive cloning in a 3-5 week time frame. When an optimal drug candidate has been selected, pools generated with EASE-containing vectors can also be used in subsequent cloning steps to make cell lines with improved expression levels. We demonstrate that EASE increases expression in nonamplified pools in addition to increasing amplification and viability of clonal cell lines generated with the EASE-containing vectors compared with pools and cell lines generated without EASE.

  17. Analysis and optimization of recombinant DNA joining reactions.

    PubMed

    Legerski, R J; Robberson, D L

    1985-01-20

    The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mM-Tris . HCl (pH 7.8), 20 mM-dithiothreitol, 10 mM-MgCl2, 1 mM-ATP) at 12 degrees C to be 1030(+/- 116) A. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.

  18. Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA.

    PubMed

    Xu, Can; Li, Zhao-Shen; Du, Yi-Qi; Tu, Zhen-Xing; Gong, Yan-Fang; Jin, Jing; Wu, Hong-Yu; Xu, Guo-Ming

    2005-01-07

    To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

  19. Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA

    PubMed Central

    Xu, Can; Li, Zhao-Shen; Du, Yi-Qi; Tu, Zhen-Xing; Gong, Yan-Fang; Jin, Jing; Wu, Hong-Yu; Xu, Guo-Ming

    2005-01-01

    AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo. PMID:15609408

  20. DNA damage tolerance by recombination: Molecular pathways and DNA structures.

    PubMed

    Branzei, Dana; Szakal, Barnabas

    2016-08-01

    Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  1. [Construction of recombinant baculovirus vectors started by EF1alpha].

    PubMed

    Gao, Dongni; Jin, Liying; Ge, Jingping; Wang, Kun; An, Qi; Ping, Wenxiang; Lou, Zhuangwei

    2014-06-04

    To improve the transduction efficiency of baculovirus and exogenous gene expression level, we chose a mammalian cell-specific promoter-human extension factor 1alpha promoter (EF1-alpha), used virus pseudotyped tools--truncated vessicular stomatitis virus protein G (VSV-GED), added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs). We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE. The recombinant transfer vectors pWK-eGFP, pWK-ITR-eGFP and pWK (-)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1alpha promoter. Constructed recombinant plasmid transfected Sf9 insect cells, and observed the expression of green fluorescent protein by using the inverted fluorescence microscope. The fluorescence expression rate of BV-WK-eGFP, BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control, ITRs can effectively extend the expression time of eGFP, the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BV-WK (-) -eGFP. The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP, BV-WK-ITR-eGFP was obviously shorten in OL cells, and reduced 24 hours compared to the negative control BV-WK (-) -eGFP, transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK (-) -eGFP, respectively. The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus, WPRE could enhance the expression efficiency of the exogenous gene significantly, and ITRs extend the expression time. The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine.

  2. Molecular genetics of DNA viruses: recombinant virus technology.

    PubMed

    Neuhierl, Bernhard; Delecluse, Henri-Jacques

    2005-01-01

    Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

  3. Construction and manipulation of giant DNA by a genome vector.

    PubMed

    Itaya, Mitsuhiro; Tsuge, Kenji

    2011-01-01

    Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.

  4. Production and titering of recombinant adeno-associated viral vectors.

    PubMed

    McClure, Christina; Cole, Katy L H; Wulff, Peer; Klugmann, Matthias; Murray, Andrew J

    2011-11-27

    In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

  5. Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

    PubMed Central

    Risseeuw, E; Franke-van Dijk, M E; Hooykaas, P J

    1996-01-01

    Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector. PMID:8816506

  6. Modulation of meiotic homologous recombination by DNA helicases.

    PubMed

    Lorenz, Alexander

    2017-05-01

    DNA helicases are ATP-driven motor proteins which translocate along DNA capable of dismantling DNA-DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR).

    PubMed

    Chandran, Sunil; Shapland, Elaine

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the yeast homologous recombination (YHR). The YHR method utilizes overlapping DNA parts that are assembled together by Saccharomyces cerevisiae via homologous recombination between designed overlapping regions. Using this method, we have successfully assembled up to 12 DNA parts in a single reaction.

  8. [Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes].

    PubMed

    Sun, Zhi-hui; Han, Jie; Shao, Lei; Wang, Li-hong; Song, Jun-xian; Wei, Zhong-hai; Zheng, Liang-rong

    2010-05-01

    To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells. The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay. The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts. The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly

  9. Reflections: ethics and the recombinant DNA debate.

    PubMed

    Robb, J W

    1982-01-01

    This essay is an attempt to apply a mode of ethical reflection to questions related to the recombinant DNA debate. The author suggests that adequate ethical analysis must include at least five aspects of a moral situation 1) the nature of the act itself; 2) consequences that result from an action or decision; 3) civil and criminal laws; 4) moral principles; and 5) motivation or intention. This paradigm for analysis is applied to the recombinant DNA scene, particularly as these factors relate to 1) risks vs benefits; 2) laws and regulations; 3) the marketplace; and 4) human genetic intervention. The analysis does not provide answers to these vexing questions, but attempts to raise the consciousness level of the reader concerning significant ethical issues. Undergirding the discussion is the humanistic theme of the primacy of the concern for the public good, and that freedom of inquiry can only survive in a socially conscious context. Fear of the unknown is no reason for prohibiting DNA research, but since the stakes are high as new developments emerge that pertain to the genetic restructuring of the human species, the author suggests that caution and responsible judgment are mandatory. Since ethics is concerned with ideal moral judgments, and applied ethics relates to the real world in which we function, the demand for clear and careful thought concerning the short-term and long-term consequences of our work is a primary ingredient of what it means to be responsible. In the final analysis, ethical responsibility rests on the individual; without a sense of personal integrity and what that implies about oneself and society, blatant opportunism can easily become the generally accepted mode of behavior and decision. If this occurs, traditional ethical concerns become irrelevant.

  10. Recombinant Mycobacterium bovis BCG as an HIV Vaccine Vector

    PubMed Central

    Chapman, Rosamund; Chege, Gerald; Shephard, Enid; Stutz, Helen; Williamson, Anna-Lise

    2011-01-01

    HIV-1 has resulted in a devastating AIDS pandemic. An effective HIV/AIDS vaccine that can be used to either, prevent HIV infection, control infection or prevent progression of the disease to AIDS is needed. In this review we discuss the use of Mycobacterium bovis BCG, the tuberculosis vaccine, as a vaccine vector for an HIV vaccine. Numerous features make BCG an attractive vehicle to deliver HIV antigens. It has a good safety profile, elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable, a necessary consideration for developing countries. In this review we discuss the numerous factors that influence generation of a genetically stable recombinant BCG vaccine for HIV. PMID:20353397

  11. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

    PubMed Central

    Condreay, J. Patrick; Witherspoon, Sam M.; Clay, William C.; Kost, Thomas A.

    1999-01-01

    Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression. PMID:9874783

  12. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  13. Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.

    PubMed

    Klymiuk, Nikolai; Fezert, Pauline; Wünsch, Annegret; Kurome, Mayuko; Kessler, Barbara; Wolf, Eckhard

    2014-05-10

    Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.

  14. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  15. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

    Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.

  16. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    PubMed

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong

    2013-07-01

    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  17. Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

    PubMed Central

    Grimm, Dirk; Pandey, Kusum; Nakai, Hiroyuki; Storm, Theresa A.; Kay, Mark A.

    2006-01-01

    We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 × 1010 to 1.8 × 1012 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV. PMID:16352567

  18. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  19. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

    PubMed

    Qi, Weicong; Tinnenbroek-Capel, Iris E M; Salentijn, Elma M J; Schaart, Jan G; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G F; Huang, Bangquan; Van Loo, Eibertus N; Krens, Frans A

    2015-09-11

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

  20. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

    PubMed Central

    Qi, Weicong; Tinnenbroek-Capel, Iris E. M.; Salentijn, Elma M. J.; Schaart, Jan G.; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G. F.; Huang, Bangquan; Van Loo, Eibertus N.; Krens, Frans A.

    2015-01-01

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera. PMID:26358007

  1. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  2. Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

    PubMed

    Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

    2016-01-01

    There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

  3. Regulation of DNA strand exchange in homologous recombination.

    PubMed

    Holthausen, J Thomas; Wyman, Claire; Kanaar, Roland

    2010-12-10

    Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Preparation of triple-block DNA polymers using recombinant DNA techniques.

    PubMed

    Selsing, E; Wells, R D

    1979-07-11

    The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described. The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase. The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E. coli. Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28. Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site. The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts. Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally.

  5. Transcript-RNA-templated DNA recombination and repair.

    PubMed

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  6. Development of a novel type of cloning vector for suicide selection of recombinants.

    PubMed

    Chan, R Y; Palfree, R G; Congote, L F; Solomon, S

    1994-03-01

    Based on the primary structure of the rat corticostatin R4 (antimicrobial defensin RatNP-1), a synthetic gene was designed, synthesized, and inserted into the IPTG-inducible prokaryotic expression vector pFLAG, with an additional 13 codons between the FLAG sequence and the synthetic R4 sequence. This construct, N-p1.2, was further developed by inclusion of multiple cloning sites right after the FLAG sequence, forming a new plasmid pSCV-1. Escherichia coli transformants containing pSCV-1 or N-p1.2 could only be propagated on agar plates in the absence of IPTG due to the detrimental expression of R4 fusion peptide to the growth of bacteria upon IPTG induction. A 214-bp bovine IGF-II cDNA and a 700-bp Ly-6C.2 cDNA fragment were subcloned into pSCV-1 and N-p1.2 respectively. Only the E. coli cells transformed with recombinant plasmids grew on IPTG agar plates. This "suicide" selection against nonrecombinants was further tested in cDNA library construction using pSCV-1. Analysis of plasmid DNA prepared from randomly picked colonies growing on ampicillin agar plates containing IPTG showed all plasmids contained cDNA inserts. The lambda Hind III fragments were used for comparing the cloning efficiency of pSCV-1 to pBluescript. Four of the 60 (6.6%) analyzed white colonies transformed with pBluescript were false positives. All of the analyzed pSCV-1-transformed colonies growing on IPTG plates contained recombinant forms of plasmid. The percentage recovery of each ligatable lambda Hind III fragment was similar in both pBluescript and pSCV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    SciTech Connect

    Not Available

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  8. Mechanics and Single-Molecule Interrogation of DNA Recombination.

    PubMed

    Bell, Jason C; Kowalczykowski, Stephen C

    2016-06-02

    The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.

  9. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  10. [Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4].

    PubMed

    Zhao, Jing; Xu, Guang-xian; Jia, Wei; Dong, Hui; Zhang, Yi-lin; Zhao, Zhi-jun; Wei, Jun

    2012-02-01

    To construct the recombined adenovirus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR- 21 with its target gene TLR4. Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  11. A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli

    PubMed Central

    Jang, Chuan-Wei; Magnuson, Terry

    2013-01-01

    Production of recombinant DNA in bacterial cells is an essential technique in molecular biology. Plasmids are usually maintained in an E. coli host by antibiotic selection. However, there are only a few antibiotic-resistance markers available in common use. Here we report the adoption of a novel selection marker, mfabI (mutant fabI) for plasmid propagation in E. coli. mfabI expands the limited repertoire of selection markers and allows for more efficient molecular manipulation and plasmid propagation in E. coli. We show that mfabI is not only an efficient plasmid selection marker, but it also possesses unique activity that may facilitate molecular manipulation of unstable sequences. Furthermore, we have incorporated mfabI in the recombineering tool kit for generating mouse gene targeting vectors and demonstrate the advantage of using mfabI-containing recombineering vectors. PMID:23437314

  12. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  13. Recombinant DNA production of spider silk proteins.

    PubMed

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

  14. 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.

  15. Single-Stranded DNA Curtains for Studying Homologous Recombination.

    PubMed

    Ma, C J; Steinfeld, J B; Greene, E C

    2017-01-01

    Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51. © 2017 Elsevier Inc. All rights reserved.

  16. Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

    PubMed

    Flotte, Terence R; Conlon, Thomas J; Poirier, Amy; Campbell-Thompson, Martha; Byrne, Barry J

    2007-03-01

    To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

  17. Advances in Non-Viral DNA Vectors for Gene Therapy

    PubMed Central

    Hardee, Cinnamon L.; Arévalo-Soliz, Lirio Milenka; Hornstein, Benjamin D.; Zechiedrich, Lynn

    2017-01-01

    Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic. PMID:28208635

  18. Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus

    PubMed Central

    Liu, Peiwen; Li, Xiaocong; Gu, Jinbao; Dong, Yunqiao; Liu, Yan; Santhosh, Puthiyakunnon; Chen, Xiaoguang

    2016-01-01

    We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control. PMID:26879823

  19. Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

    PubMed

    Liu, Peiwen; Li, Xiaocong; Gu, Jinbao; Dong, Yunqiao; Liu, Yan; Santhosh, Puthiyakunnon; Chen, Xiaoguang

    2016-02-16

    We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control.

  20. Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch.

    PubMed

    Vanoli, Fabio; Fumasoni, Marco; Szakal, Barnabas; Maloisel, Laurent; Branzei, Dana

    2010-11-11

    Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to

  1. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    SciTech Connect

    Zhao Weihong; Wu Jianqing ||; Zhong Li; Chen Linyuan; Weigel-Kelley, Kirsten A. |; Qing Keyun; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H. |; Srivastava, Arun |. E-mail: asrivastava@gtc.ufl.edu

    2006-09-30

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant

  2. Rogue athletes and recombinant DNA technology: challenges for doping control.

    PubMed

    Azzazy, Hassan M E; Mansour, Mai M H

    2007-10-01

    The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

  3. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

    PubMed Central

    Shen, Li-Zong; Wu, Wen-Xi; Xu, De-Hua; Zheng, Zhong-Cheng; Liu, Xin-Yuan; Ding, Qiang; Hua, Yi-Bing; Yao, Kun

    2002-01-01

    AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 × 1014 pfu/L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were > 15000, 216.5 ± 38.1 and 128.8 ± 25.4 μmol•L⁻¹, P < 0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the M.O.I of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 27.9 ± 4.2 μmol•L-1

  4. Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors.

    PubMed

    Hirsch, Matthew L; Wolf, Sonya J; Samulski, R J

    2016-01-01

    Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber's congenital amaurosis. In addition to rAAV's high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package "large" genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6-8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6-8, 2010; Duan et al., J Virol 73(1):161-169, 1999; Duan et al., J Virol 72(11):8568-8577, 1998; Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002). This method involves "splitting" the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002; Ghosh et al., Mol Ther 16(1):124-130, 2008; Ghosh et al., Mol Ther 15(4):750-755, 2007). The other major

  5. Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock.

    PubMed

    Davidoff, Andrew M; Ng, Catherine Y C; Sleep, Susan; Gray, John; Azam, Selina; Zhao, Yuan; McIntosh, Jenny H; Karimipoor, Morteza; Nathwani, Amit C

    2004-11-01

    Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity. The average yield of purified rAAV-8 from five different vector preparation was 41%. Electron microscopy of these purified stocks revealed typical icosohedral virions with less than 10% empty particles. Liver targeted delivery of ion-exchange purified rAAV-8 vector encoding the human factor IX (hFIX) gene, resulted in plasma hFIX levels approaching 30% of normal in immunocompetent mice, which is 20-fold higher than observed with an equivalent number of rAAV-5 ion exchange purified vector particles. The method takes less then 5 h to process and purify rAAV-8 vector from producer cells and represents a significant advance on the CsCl density centrifugation technique in current use for purification of rAAV-8 vector systems and will likely facilitate the transition of the rAAV-8 vector system to the clinic.

  6. Bioreactor production of recombinant herpes simplex virus vectors.

    PubMed

    Knop, David R; Harrell, Heather

    2007-01-01

    Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.

  7. Most Retroviral Recombinations Occur during Minus-Strand DNA Synthesis

    PubMed Central

    Zhang, Jiayou; Tang, Ling-Yun; Li, Ting; Ma, Yan; Sapp, Christy M.

    2000-01-01

    Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA. During reverse transcription, the first strand of the DNA molecule synthesized is minus-strand DNA. After the minus strand is polymerized, the plus-strand DNA is synthesized using the minus-strand DNA as the template. In this study, a helper cell line that contains two proviruses with two different mutated gfp genes was constructed. Recombination between the two frameshift mutant genes resulted in a functional gfp. If recombination occurs during minus-strand DNA synthesis, the plus-strand DNA will also contain the functional sequence. After the cell divides, all of its offspring will be green. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type gfp sequence and the minus-strand DNA will still carry the frameshift mutation. The double-stranded DNA containing this mismatch was subsequently integrated into the host chromosomal DNA of D17 cells, which were unable to repair the majority of mismatches within the retroviral double-strand DNA. After the cell divided, one daughter cell contained the wild-type gfp sequence and the other daughter cell contained the frameshift mutation in the gfp sequence. Under fluorescence microscopy, half the cells in the offspring were green and the other half of the cells were colorless or clear. Thus, we demonstrated that more than 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis. PMID:10666262

  8. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  9. Evidence for Recombination of Mitochondrial DNA in Triploid Crucian Carp

    PubMed Central

    Guo, Xinhong; Liu, Shaojun; Liu, Yun

    2006-01-01

    In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish. PMID:16322508

  10. Purification and Characterization of a DNA-Binding Recombinant PREP1:PBX1 Complex

    PubMed Central

    Mathiasen, Lisa; Bruckmann, Chiara; Pasqualato, Sebastiano; Blasi, Francesco

    2015-01-01

    Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA. PMID:25856340

  11. Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.

    PubMed

    Mathiasen, Lisa; Bruckmann, Chiara; Pasqualato, Sebastiano; Blasi, Francesco

    2015-01-01

    Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA.

  12. DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

    PubMed

    Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

    2015-12-25

    Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

  13. Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency.

    PubMed

    Farrag, Mohamed A; Amer, Haitham M; Öhlschläger, Peter; Hamad, Maaweya E; Almajhdi, Fahad N

    2017-03-08

    The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8(+) T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

  14. A wavelet-based feature vector model for DNA clustering.

    PubMed

    Bao, J P; Yuan, R Y

    2015-12-29

    DNA data are important in the bioinformatic domain. To extract useful information from the enormous collection of DNA sequences, DNA clustering is often adopted to efficiently deal with DNA data. The alignment-free method is a very popular way of creating feature vectors from DNA sequences, which are then used to compare DNA similarities. This paper proposes a wavelet-based feature vector (WFV) model, which is also an alignment-free method. From the perspective of signal processing, a DNA sequence is a sequence of digital signals. However, most traditional alignment-free models only extract features in the time domain. The WFV model uses discrete wavelet transform to adaptively yield feature vectors with a fixed dimension based on the features in both the time and frequency domains. The level of wavelet transform is adjusted according to the length of the DNA sequence rather than a fixed manually set value. The WFV model prefers a 32-dimension feature vector, which greatly promotes system performance. We compared the WFV model with the other five alignment-free models, i.e., k-tuple, DMK, TSM, AMI, and CV, on several large-scale DNA datasets on the DNA clustering application by means of the K-means algorithm. The experimental results showed that the WFV model outperformed the other models in terms of both the clustering results and the running time.

  15. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA.

    PubMed Central

    Kucherlapati, R S; Eves, E M; Song, K Y; Morse, B S; Smithies, O

    1984-01-01

    We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the frequency of this recombination can be enhanced by pretreatment of the input DNA. Two nonoverlapping deletion mutants of pSV2Neo were constructed, each affecting the bacterial aminoglycoside 3'-phosphorylase gene (the neo gene), which confers resistance to aminoglycoside antibiotics on bacteria and resistance to the antibiotic G418 on mammalian cells. Mammalian cells transfected with either deletion plasmid alone yield no G418 -resistant colonies. Cells cotransfected with both deletion plasmids yield G418 -resistant colonies with high frequency. We show that these resistant colonies result from recombination involving homologous crossing-over or gene conversion between the deletion plasmids by rescuing from the resistant cells both types of reciprocal recombinant, full-length plasmids, and doubly deleted plasmids. Cutting one of the input plasmids to generate a double-stranded gap in the neo gene considerably enhances the frequency of homologous recombination within the gene. This suggests that targeting exogenous DNA to specific sites in mammalian chromosomes could be facilitated by suitable pretreatment of the DNA. Images PMID:6328502

  16. Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

    PubMed

    Satkunanathan, Stifani; Wheeler, Jun; Thorpe, Robin; Zhao, Yuan

    2014-11-01

    Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat

  17. Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

    PubMed Central

    Satkunanathan, Stifani; Wheeler, Jun; Thorpe, Robin

    2014-01-01

    Abstract Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal

  18. Homologous recombination is required for recovery from oxidative DNA damage.

    PubMed

    Hayashi, Michio; Umezu, Keiko

    2017-04-03

    We have been studying the genetic events, including chromosome loss, chromosome rearrangements and intragenic point mutations, that are responsible for the deletion of a URA3 marker in a loss of heterozygosity (LOH) assay in the yeast Saccharomycess cerevisiae. With this assay, we previously showed that homologous recombination plays an important role in genome maintenance in response to DNA lesions that occur spontaneously in normally growing cells. Here, to investigate DNA lesions capable of triggering homologous recombination, we examined the effects of oxidative stress, a prominent cause of endogenous DNA damage, on LOH events. Treatment of log-phase cells with H2O2 first caused growth arrest and then, during the subsequent recovery, chromosome loss and various chromosome rearrangements were induced more than 10-fold. Further analysis of the rearrangements showed that gene conversion was strongly induced, approximately 100 times more frequently than in untreated cells. Consistent with these results, two diploid strains deficient for homologous recombination, rad52Δ/rad52Δ and rad51Δ/rad51Δ, were sensitive to H2O2 treatment. In addition, chromosome DNA breaks were detected in H2O2-treated cells using pulsed-field gel electrophoresis. Altogether, these results suggest that oxidative stress induced recombinogenic lesions on chromosomes, which then triggered homologous recombination leading to chromosome rearrangements, and that this response contributed to the survival of cells afflicted by oxidative DNA damage. We therefore conclude that homologous recombination is required for the recovery of cells from oxidative stress.

  19. [Construction and characterization of a novel recombinant retroviral vector expressing mouse T-bet].

    PubMed

    Zhang, Xuejie; Zhang, Jianhua; Zhang, Wei; Guo, Jie; Zhou, Xuyu

    2014-10-01

    In order to study T-bet function in mouse cells, a novel retroviral vector expressing mouse T-bet and reporter gene Thy1.1 was constructed. Retrovirus particles were then produced by transfection of the recombinant retroviral plasmid into a packaging cell line Platinum-E. The recombinant retrovirus played considerable infection ability. T-bet expression was then identified by FACS after infection of CD4+ primary T cells from T-bet knockout mouse with recombinant retrovirus. To determine if exogenous expressing T-bet has normal function, we checked the expression level of T-bet target gene, Ifng. IFN-y expression was upregulated in the T-bet knockout T cells infected with recombinant retrovirus. In conclusion, we successfully constructed an effective mouse T-bet recombinant retroviral vector.

  20. Retroviral Integrase Structure and DNA Recombination Mechanism

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2015-01-01

    SUMMARY Due to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance. PMID:25705574

  1. MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk.

    PubMed

    Monzani, Paulo S; Guemra, Samuel; Adona, Paulo R; Ohashi, Otavio M; Meirelles, Flávio V; Wheeler, Matthew B

    2015-01-01

    Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.

  2. Development of Recombinant HSV-Based Vaccine Vectors.

    PubMed

    Voellmy, Richard; Bloom, David C; Vilaboa, Nuria; Feller, Joyce

    2017-01-01

    Herpes simplex virus (HSV) causes significant morbidity on the human population through such clinical syndromes as cold sores, genital herpes, herpes stromal keratitis, and encephalitis. Attempts to generate efficacious vaccines to date have failed. We have recently described the use of a conditionally replication-competent HSV-1 vector to immunize mice against a lethal challenge of HSV-1. The unique feature of this vaccine vector is that its replication is tightly controlled and can only occur in the presence of local heat and the presence of a small molecule inducer (an antiprogestin). This gives it the safety advantage of a replication-defective vaccine vector as well as the advantage of a replication-competent vector in that it is able to stimulate innate and adaptive aspects of the immune response in a natural context that a replication-defective vector cannot. In this chapter we provide a brief overview of HSV vaccines followed by the methodology used to propagate and utilize replication-conditional HSV vectors as vaccines.

  3. Historical perspectives pertaining to the NIH Recombinant DNA Advisory Committee.

    PubMed

    Wivel, Nelson A

    2014-01-01

    Science is host to a constantly emerging series of new paradigms, and it is this characteristic that makes science both interesting and dynamic. As a part of this continuum, it became possible to create recombinant DNA molecules. Immediately it was recognized that there was a potential for serious adverse events associated with this new technology. Following two scientific conferences at Asilomar, California, the National Institutes of Health moved quickly to create the Recombinant DNA Advisory Committee (RAC). For approximately 38 years the RAC has served as an open forum for review of various recombinant DNA experiments, and for the last 23 years it has played a pivotal role in the oversight of human gene therapy. The RAC's existence obviated the need for more restrictive governmental legislation and has supported the development of genetic interventions that are leading to actual human therapies.

  4. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  5. Single Molecule Study of DNA Organization and Recombination

    NASA Astrophysics Data System (ADS)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  6. Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

    PubMed Central

    Goff, S P; Berg, P

    1978-01-01

    A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease. Images PMID:347445

  7. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    PubMed

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  8. Genome-Wide Analyses of Recombination Prone Regions Predict Role of DNA Structural Motif in Recombination

    PubMed Central

    Das, Swapan Kumar; Chowdhury, Shantanu

    2009-01-01

    HapMap findings reveal surprisingly asymmetric distribution of recombinogenic regions. Short recombinogenic regions (hotspots) are interspersed between large relatively non-recombinogenic regions. This raises the interesting possibility of DNA sequence and/or other cis- elements as determinants of recombination. We hypothesized the involvement of non-canonical sequences that can result in local non-B DNA structures and tested this using the G-quadruplex DNA as a model. G-quadruplex or G4 DNA is a unique form of four-stranded non-B DNA structure that engages certain G-rich sequences, presence of such motifs has been noted within telomeres. In support of this hypothesis, genome-wide computational analyses presented here reveal enrichment of potential G4 (PG4) DNA forming sequences within 25618 human hotspots relative to 9290 coldspots (p<0.0001). Furthermore, co-occurrence of PG4 DNA within several short sequence elements that are associated with recombinogenic regions was found to be significantly more than randomly expected. Interestingly, analyses of more than 50 DNA binding factors revealed that co-occurrence of PG4 DNA with target DNA binding sites of transcription factors c-Rel, NF-kappa B (p50 and p65) and Evi-1 was significantly enriched in recombination-prone regions. These observations support involvement of G4 DNA in recombination, predicting a functional model that is consistent with duplex-strand separation induced by formation of G4 motifs in supercoiled DNA and/or when assisted by other cellular factors. PMID:19198658

  9. Characterization of recombinant malarial RecQ DNA helicase.

    PubMed

    Suntornthiticharoen, Pattra; Srila, Witsanu; Chavalitshewinkoon-Petmitr, Porntip; Limudomporn, Paviga; Yamabhai, Montarop

    2014-08-01

    RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3' to 5' direction. The malarial RecQ1 could not unwind substrates with both 5' and 3' overhangs, those with a 5' overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity.

  10. An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

    PubMed

    Kowalczykowski, Stephen C

    2015-11-02

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  11. Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

    PubMed Central

    Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

    2014-01-01

    Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

  12. 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.

  13. 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.

  14. Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

    PubMed Central

    Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L

    2015-01-01

    The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR. PMID:25557623

  15. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  16. Eukaryotic DNA damage responses: Homologous recombination factors and ubiquitin modification.

    PubMed

    Lee, Nam Soo; Kim, Soomi; Jung, Yong Woo; Kim, Hongtae

    2017-05-06

    To prevent genomic instability disorders, cells have developed a DNA damage response. The response involves various proteins that sense damaged DNA, transduce damage signals, and effect DNA repair. In addition, ubiquitin modifications modulate the signaling pathway depending on cellular context. Among various types of DNA damage, double-stranded breaks are highly toxic to genomic integrity. Homologous recombination (HR) repair is an essential mechanism that fixes DNA damage because of its high level of accuracy. Although factors in the repair pathway are well established, pinpointing the exact mechanisms of repair and devising therapeutic applications requires more studies. Moreover, essential functions of ubiquitin modification in the DNA damage signaling pathway have emerged. In this review, to explore the eukaryotic DNA damage response, we will mention the functions of main factors in the HR repair pathway and ubiquitin modification. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  18. Preferential recombination between GC clusters in yeast mitochondrial DNA.

    PubMed Central

    Dieckmann, C L; Gandy, B

    1987-01-01

    Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome. Images Fig. 3. Fig. 4. Fig. 5. PMID:3327690

  19. The Evolution of Tandemly Repetitive DNA: Recombination Rules

    PubMed Central

    Harding, R. M.; Boyce, A. J.; Clegg, J. B.

    1992-01-01

    Variable numbers of tandem repeats (VNTRs), which include hypervariable regions, minisatellites and microsatellites, can be assigned together with satellite DNAs to define a class of noncoding tandemly repetitive DNA (TR-DNA). The evolution of TR-DNA is assumed to be driven by an unbiased recombinational process. A simulation model of unequal exchange is presented and used to investigate the evolutionary persistence of single TR-DNA lineages. Three different recombination rules are specified to govern the expansion and contraction of a TR-DNA lineage from an initial array of two repeats to, finally, a single repeat allele, which cannot participate in a misalignment and exchange process. In the absence of amplification or selection acting to bias array evolution toward expansion, the probability of attaining a target array size is a function only of the initial number of repeats. We show that the proportions of lineages attaining a targeted array size are the same irrespective of recombination rule and rate, demonstrating that our simulation model is well behaved. The time taken to attain a target array size, the persistence of the target array, and the total persistence time of repetitive array structure, are functions of the initial number of repeats, the rate of recombination, and the rules of misalignment preceding recombinational exchange. These relationships are investigated using our simulation model. While misalignment contraint is probably greatest for satellite DNA it also seems important in accounting for the evolution of VNTR loci including minisatellites. This conclusion is consistent with the observed nonrandom distributions of VNTRs and other TR-DNAs in the human genome. PMID:1468634

  20. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

    PubMed

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu

    2016-08-01

    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights

  1. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  2. A Collaborative, Investigative Recombinant DNA Technology Course with Laboratory

    ERIC Educational Resources Information Center

    Pezzementi, Leo; Johnson, Joy F.

    2002-01-01

    A recombinant DNA technology course was designed to promote contextual, collaborative, inquiry-based learning of science where students learn from one another and have a sense of ownership of their education. The class stressed group presentations and critical reading and discussion of scientific articles. The laboratory consisted of two research…

  3. A Collaborative, Investigative Recombinant DNA Technology Course with Laboratory

    ERIC Educational Resources Information Center

    Pezzementi, Leo; Johnson, Joy F.

    2002-01-01

    A recombinant DNA technology course was designed to promote contextual, collaborative, inquiry-based learning of science where students learn from one another and have a sense of ownership of their education. The class stressed group presentations and critical reading and discussion of scientific articles. The laboratory consisted of two research…

  4. The influenza virus nucleoprotein synthesized from cloned DNA in a simian virus 40 vector is detected in the nucleus.

    PubMed Central

    Lin, B C; Lai, C J

    1983-01-01

    We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza A virus by reverse transcription of virion RNA with synthetic oligonucleotide primers. Terminal sequence analysis showed that the cloned gene contained a full-length copy of the virion RNA segment. The NP-specific DNA was inserted into the late region of a simian virus 40 vector, and the DNA recombinant was propagated in the presence of an early simian virus 40 temperature-sensitive mutant helper. Infection of African green monkey kidney cells with the recombinant produced a polypeptide immunoprecipitable with NP-specific antisera. The polypeptide product had a molecular weight of 56,000, identical to that of the nucleoprotein of influenza virus as estimated on polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection. Images PMID:6296449

  5. Recombination of mitochondrial DNA detected in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy

    PubMed Central

    Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E.; Khrapko, Konstantin; Kunz, Wolfram S.

    2006-01-01

    Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in one exceptional individual with paternal inheritance of mtDNA1 and in an in vitro cell culture system2. Whether mtDNA recombination is a common event in humans remained to be elucidated. To detect mtDNA recombination in human skeletal muscle, we have analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In ten out of ten individuals who harbored a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Reassuringly, 12 out of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle. PMID:16025113

  6. [Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene].

    PubMed

    Ding, Zhi-xiang; Tan, Qian; Liu, Shuang-zhen; Liu, Dan; Li, Zhong-qing; Peng, Jian-qiang

    2008-03-01

    To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.

  7. DNA sequence alignment by microhomology sampling during homologous recombination.

    PubMed

    Qi, Zhi; Redding, Sy; Lee, Ja Yil; Gibb, Bryan; Kwon, YoungHo; Niu, Hengyao; Gaines, William A; Sung, Patrick; Greene, Eric C

    2015-02-26

    Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair single-strand DNA (ssDNA) with a homologous double-strand DNA (dsDNA) template. Here, we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a ninth nucleotide coincides with an additional reduction in binding free energy, and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms*

    PubMed Central

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-01-01

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. PMID:25170075

  9. Filter replicas and permanent collections of recombinant DNA plasmids.

    PubMed Central

    Gergen, J P; Stern, R H; Wensink, P C

    1979-01-01

    A permanent, ordered collection of 23,000 recombinant DNA plasmids containing Drosophila melanogaster DNA has been established. Simple and practical methods for storing and manipulating this collection were developed. In addition, an improved, simple and inexpensive method for making paper filter replicas of such an ordered collection and of a high density (10,000 colonies/petri dish) unordered collection was developed. These filter replicas are suitable for nucleic acid hybridization screens of recombinant DNA colinies and each filter replica can be used for many (greater than 5) successive screens. The kinetics of this hybridization reaction were examined and allow design of experiments that detect colony complementarity to a nucleic acid that is 0.5% of the hybridization probe. Images PMID:118435

  10. Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

    PubMed

    Burnham, Brenda; Nass, Shelley; Kong, Elton; Mattingly, MaryEllen; Woodcock, Denise; Song, Antonius; Wadsworth, Samuel; Cheng, Seng H; Scaria, Abraham; O'Riordan, Catherine R

    2015-12-01

    Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

  11. [The applications of thermostable ligase chain reaction in facilitating DNA recombination].

    PubMed

    Xiangda, Zhou; Xiao, Song; Cong, Huai; Haiyan, Sun; Hongyan, Chen; Daru, Lu

    2016-02-01

    The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

  12. Recombinant vesicular stomatitis virus vector mediates postexposure protection against Sudan Ebola hemorrhagic fever in nonhuman primates.

    PubMed

    Geisbert, Thomas W; Daddario-DiCaprio, Kathleen M; Williams, Kinola J N; Geisbert, Joan B; Leung, Anders; Feldmann, Friederike; Hensley, Lisa E; Feldmann, Heinz; Jones, Steven M

    2008-06-01

    Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.

  13. Role of Recombinant DNA Technology to Improve Life

    PubMed Central

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life. PMID:28053975

  14. Role of Recombinant DNA Technology to Improve Life.

    PubMed

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad; Hou, Hongwei

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life.

  15. [Gene expression of AAV-ITR ssDNA mini vector in skeletal muscle of mice].

    PubMed

    Zhu, Dongqin; Zhang, Yun; Liu, Xiaomei; Zhang, Chun

    2014-11-01

    AAV-ITR single strand DNA mini vector (AAV-ITR ssDNA mini vector) is a novel gene expression vector based on AAV-ITR. We have shown efficient gene expression of AAV-ITR ssDNA mini vector in HEK 293T. Here, we studied the efficacy of gene expression of AAV-ITR ssDNA mini vector in vivo. We injected the skeletal muscle of ICR mice separately with equal molars of AAV-ITR ssDNA mini vector, ITR mutated AAV-ITR single strand DNA mini vector (AAV-ITRmm ssDNA mutant vector), AAV-ITR dsDNA and pUC57-minivector-GFP, combined with TurboFect. Florescence microscope analysis of skeletal muscle section shows that AAV-ITR ssDNA mini vector had higher expression efficiency and longer expression period. We extracted DNA from the muscle three months after injection and quantified three vectors by Real-time PCR. RT-PCR analysis shows that there were highest copy numbers of AAV-ITR ssDNA mini vector existing in muscle. Stable existing of AAV- TR ssDNA mini vector in muscle could be the molecular basis of long term gene expression of the vector. The results suggest that AAV-ITR ssDNA mini vector might be a promising vector for gene therapy.

  16. High density recombinant AAV particles are competent vectors for in vivo transduction

    USDA-ARS?s Scientific Manuscript database

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  17. [The construction of attenuated Tiantan recombinant vaccinia virus vector with IFN-gamma receptor gene deletion].

    PubMed

    Huang, Wei; Liu, Ying; Duan, Dan-li; Li, Hai-shan; Liu, Yong; Hong, Kun-Xue; Zhu, Jia-hong; Shao, Yi-ming

    2004-03-01

    B8R gene encodes a secreted protein with homology to IFN-gamma receptor, which neutralizes the antiviral and immunological regulation activities of IFN-gamma. To improve the safety of vaccinia virus vector, an attenuated recombinant vaccinia virus with the B8R gene deletion from Tiantan vaccine strain (VTT) was constructed. The transfer vectors were generated by joining B8R left flank, B8R right flank, vv promoter, LacZ, multicloning site and pBRSK fragments. The recombinant viruses VTTdeltaB8RLacZ (VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination. The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTdeltaB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTdeltaB8RLacZ (10(6) pfu) were significantly smaller and healed faster than those formed by VTT when injected intradermally to the rabbits,and no visible ulceration occurred. Meanwhile LacZ in VTKgpedeltaB8RLacZ was expressed stably. The attenuated vector with B8R gene deletion improves the safety of recombinant vaccinia virus vaccine B8R locus may be used as a new site for insertion of foreign genes in vaccinia virus vector.

  18. Recombinant viral vectored vaccines for the control of avian influenza: a review

    USDA-ARS?s Scientific Manuscript database

    The poultry industry has been at the forefront of developing recombinant viral vectored vaccines in an attempt to improve the immune response to vaccination. With AIV, the hemagglutinin surface glycoprotein is the key antigen for protection against infection. This allows a single gene to be transf...

  19. 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.

  20. Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors

    PubMed Central

    Hirsch, Matthew L.; Wolf, Sonya J.; Samulski, R.J.

    2016-01-01

    Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber’s congenital amaurosis. In addition to rAAV’s high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package “large” genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6–8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6–8, 2010; Duan et al., J Virol 73(1):161–169, 1999; Duan et al., J Virol 72(11):8568–8577, 1998; Duan et al., Mol Ther 4(4):383–391, 2001; Halbert et al., Nat Biotechnol 20(7):697–701, 2002). This method involves “splitting” the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383–391, 2001; Halbert et al., Nat Biotechnol 20(7):697–701, 2002; Ghosh et al., Mol Ther 16(1):124–130, 2008; Ghosh et al., Mol Ther 15

  1. Endonucleases involved in repair and recombination of DNA

    SciTech Connect

    Linn, S.M.

    1988-01-01

    When our DOE support began as a contract in 1970, from the AEC, it was our intent to begin to understand how several enzymes which we had detected in E. coli might be involved in DNA recombination and repair. These studies led to our characterization of the recBC DNase (exonuclease 5) as well as endonucleases 3 and 5. As research supported by that contract progressed, we expanded our interests to include mammalian enzymes involved in base excision repair, most notably AP endonucleases, DNA glycosylases and DNA purine insertase. A logical next step involved the inclusion of DNA polymerases into our studies of repair. Current progress includes research on: isolation of xeroderma pigmentosum correction factors; isolation of ultraviolet (UV) endonucleases; mitochondrial repair enzymes; alkylation damage repair; comparisons of repair in normal diploid, transformed, and non-mitotic cells; and repair reactions by DNA polymerases.

  2. Exploration of BAC versus plasmid expression vectors in recombinant CHO cells.

    PubMed

    Mader, Alexander; Prewein, Bernhard; Zboray, Katalin; Casanova, Emilio; Kunert, Renate

    2013-05-01

    Vector engineering approaches are commonly used to increase recombinant protein production in mammalian cells, and among various concepts, bacterial artificial chromosomes (BAC) have been proposed to serve as open chromatin regions to omit chromosome positional effects. For proof of concept, we developed stable recombinant Chinese hamster ovary (CHO) cell lines using different expression vector systems: the plasmid vectors contained the identical expression cassette as the BAC constructs. Two anti-HIV1 antibody derivates served as model proteins (3D6scFc and 2F5scFc) for generation of four stable recombinant CHO cell lines. The BAC-derived clones showed three to four times higher specific productivity, and therefore, gene copy numbers and transcript level were quantified. The active chromatin region provided with the BAC environment significantly improved transcription evidenced with both model proteins. Specific transcription was approximately six times higher from BAC-based vectors compared to the corresponding plasmid vectors for both single-chain fragment crystallizable (scFc) proteins. Our accurate investigations elucidated also differences between translational activities related to the protein of choice. 3D6scFc expressed specifically three to four times more product than 2F5scFc indicating that the product by itself also contributes to enhanced productivity. This study indicated comparable increase of transcription level for both scFc proteins when using the BAC system, but translation, maturation, and secretion of individual proteins seem to be protein specific.

  3. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... ] under Section III-A-1 of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... the NIH Recombinant DNA Advisory Committee has been deferred at the request of the principal...

  4. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery.

    PubMed

    Wang, Qizhao; Dong, Biao; Firrman, Jenni; Roberts, Sean; Moore, Andrea Rossi; Cao, Wenjing; Diao, Yong; Kapranov, Philipp; Xu, Ruian; Xiao, Weidong

    2014-08-01

    Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (∼5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

  5. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    PubMed Central

    Najor, Nicole A.; Weatherford, Layne; Brush, George S.

    2016-01-01

    In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control. PMID:27678521

  6. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

  7. Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

    PubMed Central

    Schwarzenberger, P; Hunt, J D; Robert, E; Theodossiou, C; Kolls, J K

    1997-01-01

    To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA. PMID:9343214

  8. Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses.

    PubMed

    Santra, Sampa; Barouch, Dan H; Korioth-Schmitz, Birgit; Lord, Carol I; Krivulka, Georgia R; Yu, Faye; Beddall, Margaret H; Gorgone, Darci A; Lifton, Michelle A; Miura, Ayako; Philippon, Valerie; Manson, Kelledy; Markham, Phillip D; Parrish, John; Kuroda, Marcelo J; Schmitz, Jörn E; Gelman, Rebecca S; Shiver, John W; Montefiori, David C; Panicali, Dennis; Letvin, Norman L

    2004-07-27

    Although a consensus has emerged that an HIV vaccine should elicit a cytotoxic T lymphocyte (CTL) response, the characteristics of an effective vaccine-induced T lymphocyte response remain unclear. We explored this issue in the simian human immunodeficiency virus/rhesus monkey model in the course of assessing the relative immunogenicity of vaccine regimens that included a cytokine-augmented plasmid DNA prime and a boost with DNA or recombinant pox vectors. Recombinant vaccinia virus, recombinant modified vaccinia Ankara (MVA), and recombinant fowlpox were comparable in their immunogenicity. Moreover, whereas the magnitude of the peak vaccine-elicited T lymphocyte responses in the recombinant pox virus-boosted monkeys was substantially greater than that seen in the monkeys immunized with plasmid DNA alone, the magnitudes of recombinant pox boosted CTL responses decayed rapidly and were comparable to those of the DNA-alone-vaccinated monkeys by the time of viral challenge. Consistent with these comparable memory T cell responses, the clinical protection seen in all groups of experimentally vaccinated monkeys was similar. This study, therefore, indicates that the steady-state memory, rather than the peak effector vaccine-elicited T lymphocyte responses, may be the critical immune correlate of protection for a CTL-based HIV vaccine.

  9. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

    PubMed

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-10-01

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  10. Recombinant DNA in Japan: current status and future prospects

    SciTech Connect

    Not Available

    1989-01-01

    The goals of the report are to evaluate the current status of Japanese Recombinant DNA Biotechnology, and to suggest ways to improve the use of the Japanese biotechnology literature. Abstracts and titles of papers presented at Japanese scientific meetings held from November 1987 to November 1988 were evaluated and translated to give the reader an overall idea of the areas in which Japanese researchers are active. In general, Japanese recombinant DNA technology is on a par with that in the U.S. - there is no technology lead on either side. The author recommends that U.S. bio-researchers should read the Japanese language literature, particularly in applied areas, since the abstracts of meetings held in Japan in Japanese are a good source of current, concise information.

  11. Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells.

    PubMed

    Kondratov, Oleksandr; Marsic, Damien; Crosson, Sean M; Mendez-Gomez, Hector R; Moskalenko, Oleksandr; Mietzsch, Mario; Heilbronn, Regine; Allison, Jonathan R; Green, Kari B; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2017-08-10

    The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  12. Vector Design for Improved DNA Vaccine Efficacy, Safety and Production

    PubMed Central

    Williams, James A.

    2013-01-01

    DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy. PMID:26344110

  13. Single-polarity recombinant adeno-associated virus 2 vector-mediated transgene expression in vitro and in vivo: mechanism of transduction.

    PubMed

    Zhong, Li; Zhou, Xiaohuai; Li, Yanjun; Qing, Keyun; Xiao, Xiao; Samulski, Richard Jude; Srivastava, Arun

    2008-02-01

    Recombinant adeno-associated virus 2 (AAV) vectors encapsidate single-stranded genomes of either polarity equally frequently in separate mature virions. Because viral genomes of either polarity are transcriptionally inactive, both the failure to undergo viral second-strand DNA synthesis and the failure to undergo DNA strand annealing have been proposed as possible reasons to account for the observed low efficiency of transgene expression. We compared the transduction efficiencies of conventional AAV vectors containing both [-] and [+] polarity genomes with those containing either the [-] or the [+] polarity genomes, in vitro as well as in vivo. We document that the transduction efficiency of single-polarity AAV vectors is significantly enhanced by (i) co-infection with adenovirus; (ii) small interfering RNA (siRNA)-mediated down-modulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which inhibit AAV second-strand DNA synthesis; (iii) over-expression of a cellular protein tyrosine phosphatase, T cell protein tyrosine phosphatase (TC-PTP), which catalyzes tyrosine-dephosphorylation of FKBP52; and (iv) deliberate over-expression of TC-PTP, or the absence of FKBP52, respectively, in TC-PTP-transgenic mice and in FKBP52-knockout mice. These data confirm that viral second-strand DNA synthesis, rather than DNA strand annealing, is the rate-limiting step in efficient transduction by AAV vectors. This finding has implications in the use of these vectors in human gene therapy.

  14. Jeremy Rifkin challenges recombinant DNA research: A rhetoric of heresy

    SciTech Connect

    Futrell, W.M.

    1992-01-01

    One significant issue to come before the public in recent years is recombinant DNA research or genetic engineering and its applications. An important spokesman on this issue is Jeremy Rifkin. Rifkin is of rhetorical interest because of his strategies to sustain the dialogue and define the parameters in which it occurs. This dissertation analyzes a broad range of Rifkin's rhetorical artifacts and those of scientists engaged in recombinant DNA research. They are examined against criteria developed to identify and understand heresy. The five areas of analysis are: the nearness/remoteness phenomenon, the social construction of heresy, the social consequences of heresy, the doctrinal consequences of heresy, and the heresy-hunt ritual. The first two criteria focus on the rhetorical strategies of the heretic. The last three concentrate on the rhetorical strategies of the defenders of the institutional orthodoxy. This dissertation examines the rhetorical strategies of a heretical challenge to the scientific establishment and the consequences of that challenge. This dissertation also analyzes the rhetorical strategies employed by the defenders of the scientific orthodoxy. Although an understanding of the rhetorical strategies employed on both sides of this conflict is important, the implications for the role of rhetoric in highly controversial issues such as recombinant DNA are even more critical.

  15. Physical studies of chromatin. The recombination of histones with DNA.

    PubMed

    Boseley, P G; Bradbury, E M; Butler-Browne, G S; Carpenter, B G; Stephens, R M

    1976-02-02

    Experiments have been carried out to define clearly which histone combinations can induce a higher order structure when combined with DNA. The criterion for a higher order structure being the series of low-angle X-ray diffraction maxima nominally at 5.5 nm, 3.7 nm, 2.7 nm and 2.2 nm. Such a pattern, with resolution similar to that of H1-depleted chromatin, is readily attainable by recombining histones H2A + H2B + H3 + H4 with DNA using a salt-gradient dialysis method. However, the use of urea in the recombination procedure is shown to be detrimental to the production of a higher order structure. Low-angle ring patterns are not obtained by recomgining DNA with single pure histones or any combination of histone pairs exept H3 + H4. The diffraction maxima from the latter are, however, weaker than those from chromatin and there are pronounced semi-equatorial arcs. The presence of a third histone, either H2A or H2B in the H3 + H4 recombination mixture tends to distort the recognised low-angle pattern. It is concluded that the histone pair H3 + H4 is essential for the formation of a regular higher order structure in chromatin, although for a complete structural development the presence of H2A + H2B is also required.

  16. Lentiviral Vectors for the Engineering of Implantable Cells Secreting Recombinant Antibodies.

    PubMed

    Lathuilière, Aurélien; Schneider, Bernard L

    2016-01-01

    The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody.

  17. Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge

    PubMed Central

    Matassov, Demetrius; Seymour, Robert L.; Latham, Theresa; Gorchakov, Rodion V.; Nowak, Rebecca M.; Leal, Grace; Hamm, Stefan; Eldridge, John H.; Tesh, Robert B.; Clarke, David K.

    2017-01-01

    ABSTRACT The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans. IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector

  18. Protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines.

    PubMed

    Johnson, Deirdre I; Vagnozzi, Ariel; Dorea, Fernanda; Riblet, Sylva M; Mundt, Alice; Zavala, Guillermo; García, Maricarmen

    2010-12-01

    Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines. The chicken embryo origin (CEO) vaccines, although proven to be effective in experimental settings, have limited efficacy in controlling the disease in dense broiler production sites due to unrestricted use and poor mass vaccination coverage. These factors allowed CEO vaccines to regain virulence, causing long lasting and, consequently, severe outbreaks of the disease. A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The objective of this study was to evaluate the protection induced by ILTV viral vector recombinant vaccines after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. In this study, both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.

  19. Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

    SciTech Connect

    Kaur, Amitinder . E-mail: amitinder_kaur@hms.harvard.edu; Sanford, Hannah B.; Garry, Deirdre; Lang, Sabine; Klumpp, Sherry A.; Watanabe, Daisuke; Bronson, Roderick T.; Lifson, Jeffrey D.; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Knipe, David M.; Desrosiers, Ronald C.

    2007-01-20

    The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value < 0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value < 0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.

  20. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

    PubMed

    D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  1. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    PubMed Central

    D’Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field. PMID:27069952

  2. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

    PubMed Central

    Kogoma, T

    1997-01-01

    Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored. PMID:9184011

  3. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

    PubMed

    Harding, C O; Gillingham, M B; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, D D

    2006-03-01

    Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

  4. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

    PubMed Central

    Harding, CO; Gillingham, MB; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, DD

    2009-01-01

    Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pahenu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5±2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  5. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    SciTech Connect

    Not Available

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro.

  6. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination

    PubMed Central

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-01-01

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8+ T cells but by specific antibodies. Finally, by using C3−/− mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way. PMID:26667202

  7. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination.

    PubMed

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-12-15

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8(+) T cells but by specific antibodies. Finally, by using C3(-/-) mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way.

  8. Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

    PubMed

    Chakrabarti, S; Joffe, S; Seidman, M M

    1985-09-01

    Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.

  9. Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

    PubMed Central

    Chakrabarti, S; Joffe, S; Seidman, M M

    1985-01-01

    Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools. Images PMID:3869955

  10. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  11. Choreography of recombination proteins during the DNA damage response.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2009-09-02

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.

  12. Multivalent dendrimer vectors with DNA intercalation motifs for gene delivery.

    PubMed

    Wong, Pamela T; Tang, Kenny; Coulter, Alexa; Tang, Shengzhuang; Baker, James R; Choi, Seok Ki

    2014-11-10

    Poly(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and with 3,8-diamino-6-phenylphenanthridinium ("DAPP")-derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon coincubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: KD = 0.22 nM for G5(FA)(DAPP) versus 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line and showed that RF-conjugated dendrimers were 1 to 2 orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery.

  13. Limited infection upon human exposure to a recombinant raccoon pox vaccine vector.

    PubMed

    Rocke, Tonie E; Dein, F Joshua; Fuchsberger, Martina; Fox, Barry C; Stinchcomb, Dan T; Osorio, Jorge E

    2004-07-29

    A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

  14. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period.

  15. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    PubMed

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  16. The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper.

    PubMed

    Pan, Zihao; Liu, Jin; Ma, Jiale; Jin, Qiuli; Yao, Huochun; Osterrieder, Nikolaus

    2017-10-01

    Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper. Copyright © 2017. Published by Elsevier Ltd.

  17. Unique safety issues associated with virus-vectored vaccines: Potential for and theoretical consequences of recombination with wild type virus strains.

    PubMed

    Condit, Richard C; Williamson, Anna-Lise; Sheets, Rebecca; Seligman, Stephen J; Monath, Thomas P; Excler, Jean-Louis; Gurwith, Marc; Bok, Karin; Robertson, James S; Kim, Denny; Michael Hendry, R; Singh, Vidisha; Mac, Lisa M; Chen, Robert T

    2016-12-12

    In 2003 and 2013, the World Health Organization convened informal consultations on characterization and quality aspects of vaccines based on live virus vectors. In the resulting reports, one of several issues raised for future study was the potential for recombination of virus-vectored vaccines with wild type pathogenic virus strains. This paper presents an assessment of this issue formulated by the Brighton Collaboration. To provide an appropriate context for understanding the potential for recombination of virus-vectored vaccines, we review briefly the current status of virus-vectored vaccines, mechanisms of recombination between viruses, experience with recombination involving live attenuated vaccines in the field, and concerns raised previously in the literature regarding recombination of virus-vectored vaccines with wild type virus strains. We then present a discussion of the major variables that could influence recombination between a virus-vectored vaccine and circulating wild type virus and the consequences of such recombination, including intrinsic recombination properties of the parent virus used as a vector; sequence relatedness of vector and wild virus; virus host range, pathogenesis and transmission; replication competency of vector in target host; mechanism of vector attenuation; additional factors potentially affecting virulence; and circulation of multiple recombinant vectors in the same target population. Finally, we present some guiding principles for vector design and testing intended to anticipate and mitigate the potential for and consequences of recombination of virus-vectored vaccines with wild type pathogenic virus strains. Copyright © 2016. Published by Elsevier Ltd.

  18. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

    PubMed

    Kuzminov, A

    2001-07-17

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

  19. Reducing the immunostimulatory activity of CpG-containing plasmid DNA vectors for non-viral gene therapy.

    PubMed

    Yew, Nelson S; Cheng, Seng H

    2004-11-01

    The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid-DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.

  20. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

    SciTech Connect

    Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi; Mathews, Lydia; Lucas, William T.; Murphy, Cynthia G.; Felber, Barbara K.; Pavlakis, George N.; Deluca, Neal A.; Knipe, David M. . E-mail: david_knipe@hms.harvard.edu

    2007-01-20

    Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.

  1. Process of labeling specific chromosomes using recombinant repetitive DNA

    DOEpatents

    Moyzis, R.K.; Meyne, J.

    1988-02-12

    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 members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

  2. Differential requirements of singleplex and multiplex recombineering of large DNA constructs.

    PubMed

    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

    2015-01-01

    Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.

  3. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  4. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  5. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  6. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  7. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  8. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    PubMed

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  9. DNA transformations of Candida tropicalis with replicating and integrative vectors.

    PubMed

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

  10. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

    PubMed Central

    Lecomte, Emilie; Tournaire, Benoît; Cogné, Benjamin; Dupont, Jean-Baptiste; Lindenbaum, Pierre; Martin-Fontaine, Mélanie; Broucque, Frédéric; Robin, Cécile; Hebben, Matthias; Merten, Otto-Wilhelm; Blouin, Véronique; François, Achille; Redon, Richard; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles. PMID:26506038

  11. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.

    PubMed

    Lecomte, Emilie; Tournaire, Benoît; Cogné, Benjamin; Dupont, Jean-Baptiste; Lindenbaum, Pierre; Martin-Fontaine, Mélanie; Broucque, Frédéric; Robin, Cécile; Hebben, Matthias; Merten, Otto-Wilhelm; Blouin, Véronique; François, Achille; Redon, Richard; Moullier, Philippe; Léger, Adrien

    2015-10-27

    Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.

  12. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells.

    PubMed

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-03-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.

  13. Relevance of a pre-existing measles immunity prior immunization with a recombinant measles virus vector.

    PubMed

    Knuchel, Marlyse C; Marty, René R; Morin, Teldja Neige Azzouz; Ilter, Orhan; Zuniga, Armando; Naim, Hussein Y

    2013-03-01

    Measles virus (MV) vectors are promising candidates for designing new recombinant vaccines since the parental live vaccines have a well-known safety and efficacy record. Like all viral vectors, the MV vector efficacy in inducing a protecting immune answer could be affected by the pre-existing immunity among the human population. In order to determine the optimal immunization route and regimen, we mimicked a MV pre-immunity by passively administrating MV neutralizing antibodies (MV-nAb) prior intramuscular (i.m.) and/or intranasal (i.n.) immunization with recombinant MV expressing the SIV-gag antigen (rMV-SIVgag). Our results revealed that 500 mIU of MV-nAb allowed the induction of a humoral and cellular immune response against the vector and the transgene, while higher titers of the MV-nAb were significantly inhibitory. In a prime-boost regimen, in the presence of MV-nAb, the intranasal-intramuscular (i.n.-i.m.) or intramuscular-intramuscular (i.m.-i.m.) routes induced higher humoral immune responses against the vector and the transgene (SIV-gag). In naive animals, cellular immune response was significantly higher by i.m. immunization; however, MV pre-immunity did not seem to affect the cellular immune response after an i.n. immunization.   In summary, we show that a pre-existing immunity of up to 500 mIU anti-MV neutralizing antibodies had little effect on the replication of rMV and did not inhibit the induction of significant humoral and cellular immune responses in immune-competent mice.

  14. [Construction of recombinate luminescence bacteria vector to evaluate the genetoxic of environment pollutant].

    PubMed

    Huang, Xin-Xin; He, Miao; Shi, Han-Chang; Cai, Qiang

    2008-11-01

    Recombinate luminescence bacteria have the important role in evaluating water toxicity. Two recombinate luminescence bacteria vectors PUCD-uvrA and PUCD-alkA were constructed to investigate the impaired mechanism of pollutant genetic toxicity. The genes of uvrA and alkA were amplified by PCR from E. coli W3110, sequenced after ligated with pGEM-T easy vector. The PCR products and PUCD615 vector were all digested with BamH I, EcoR I, then be linked and imported into JM109 with electrotransformation. Several clones were selected and identificated by PCR and sequencing. The results reviewed that the length of the uvrA and the alkA fragments were 237 bp, 326 bp. When they were sequenced and blasted in GenBank, the homology of sequences reached 99% indicated the amplified results correct. The results of sequencing ligated with PUCD615 reviewed that the fragments of uvrA and alkA had been inserted into the multiple clone site correctly, the insert direction and reading frame were also exactly. Optimizing the condition of ligation and transformation, the large fragment of PUCD615 and the short inserted sequences can been ligated successfully.

  15. Phylogenetic inference of Indian malaria vectors from multilocus DNA sequences.

    PubMed

    Dixit, Jyotsana; Srivastava, Hemlata; Sharma, Meenu; Das, Manoj K; Singh, O P; Raghavendra, K; Nanda, Nutan; Dash, Aditya P; Saksena, D N; Das, Aparup

    2010-08-01

    Inferences on the taxonomic positions, phylogenetic interrelationships and divergence time among closely related species of medical importance is essential to understand evolutionary patterns among species, and based on which, disease control measures could be devised. To this respect, malaria is one of the important mosquito borne diseases of tropical and sub-tropical parts of the globe. Taxonomic status of malaria vectors has been so far documented based on morphological, cytological and few molecular genetic features. However, utilization of multilocus DNA sequences in phylogenetic inferences are still in dearth. India contains one of the richest resources of mosquito species diversity but little molecular taxonomic information is available in Indian malaria vectors. We herewith utilized the whole genome sequence information of An. gambiae to amplify and sequence three orthologous nuclear genetic regions in six Indian malaria vector species (An. culicifacies, An. minimus, An. sundaicus, An. fluviatilis, An. annularis and An. stephensi). Further, we utilized the previously published DNA sequence information on the COII and ITS2 genes in all the six species, making the total number of loci to five. Multilocus molecular phylogenetic study of Indian anophelines and An. gambiae was conducted at each individual genetic region using Neighbour Joining (NJ), Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian approaches. Although tree topologies with COII, and ITS2 genes were similar, for no other three genetic regions similar tree topologies were observed. In general, the reconstructed phylogenetic status of Indian malaria vectors follows the pattern based on morphological and cytological classifications that was reconfirmed with COII and ITS2 genetic regions. Further, divergence times based on COII gene sequences were estimated among the seven Anopheles species which corroborate the earlier hypothesis on the radiation of different species of the Anopheles

  16. Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

    PubMed Central

    Binet, Rachel; Maurelli, Anthony T.

    2009-01-01

    To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 × 10−9). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 μg of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 ± 1.1 × 10−6, n = 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia. PMID:19104068

  17. Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2009-01-06

    To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 x 10(-9)). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 microg of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 +/- 1.1 x 10(-6), n = 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.

  18. Recombination promoted by DNA viruses: phage λ to herpes simplex virus.

    PubMed

    Weller, Sandra K; Sawitzke, James A

    2014-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

  19. Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

    PubMed Central

    Weller, Sandra K.; Sawitzke, James A.

    2015-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

  20. Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability.

    PubMed

    Nowosielska, Anetta

    2007-01-01

    Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.

  1. Statistical Analysis on Detecting Recombination Sites in DNA-β Satellites Associated with Old World Geminiviruses

    PubMed Central

    Xu, Kai; Yoshida, Ruriko

    2010-01-01

    Although exchange of genetic information by recombination plays an important role in the evolution of viruses, it is not clear how it generates diversity. Understanding recombination events helps with the study of the evolution of new virus strains or new viruses. Geminiviruses are plant viruses which have ambisense single-stranded circular DNA genomes and are one of the most economically important plant viruses in agricultural production. Small circular single-stranded DNA satellites, termed DNA-β, have recently been found to be associated with some geminivirus infections. In this paper we analyze several DNA-β sequences of geminiviruses for recombination events using phylogenetic and statistical analysis and we find that one strain from ToLCMaB has a recombination pattern and is a recombinant molecule between two strains from two species, PaLCuB-[IN:Chi:05] (major parent) and ToLCB-[IN:CP:04] (minor parent). We propose that this recombination event contributed to the evolution of the strain of ToLCMaB in South India. The Hidden Markov Chain (HMM) method developed by Webb et al. (2009) estimating phylogenetic tree through out the whole alignment provide us a recombination history of these DNA-β strains. It is the first time that this statistic method has been used on DNArecombination study and give a clear recombination history of DNArecombination. PMID:21423447

  2. A universal vector for high-efficiency multi-fragment recombineering of BACs and knock-in constructs.

    PubMed

    Dolt, Karamjit Singh; Lawrence, Melanie L; Miller-Hodges, Eve; Slight, Joan; Thornburn, Anna; Devenney, Paul S; Hohenstein, Peter

    2013-01-01

    There is an increasing need for more efficient generation of transgenic constructs. Here we present a universal multi-site Gateway vector for use in recombineering reactions. Using transgenic mouse models, we show its use for the generation of BAC transgenics and targeting vectors. The modular nature of the vector allows for rapid modification of constructs to generate different versions of the same construct. As such it will help streamline the generation of series of related transgenic models.

  3. A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells.

    PubMed

    Bouabe, Hicham; Okkenhaug, Klaus

    2013-01-01

    The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem cells (ESCs) that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g., insertion of human-specific genes or reporter genes), gene disruption, point mutations, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

  4. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures

    SciTech Connect

    Chen, Zhucheng; Yang, Haijuan; Pavletich, Nikola P

    2008-07-08

    The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP {gamma}-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

  5. Herpes simplex virus clearance during purification of a recombinant adeno-associated virus serotype 1 vector.

    PubMed

    Ye, Guo-jie; Scotti, Marina M; Thomas, Darby L; Wang, Lijun; Knop, David R; Chulay, Jeffrey D

    2014-12-01

    Gene delivery vectors based on adeno-associated virus (AAV) have potential utility for treatment of many genetic disorders. Current AAV vector manufacturing methods employ helper viruses to deliver functions needed to produce replication-defective recombinant AAV (rAAV) vectors, and clearance of infectious helper virus from the drug substance is essential for ensuring the safety of rAAV-based therapies. We have developed a manufacturing method for the production of rAAV vectors using a recombinant herpes simplex virus type 1 (rHSV) complementation system in suspension baby hamster kidney cells. The manufacturing process includes three primary unit operations, detergent lysis of the cell harvest and two downstream column chromatography steps, which achieve viral clearance. These unit operations inactivate and remove HSV, including replication-competent HSV present at low levels in rHSV helper stocks. Here we report results quantifying the reduction in HSV achieved during rAAV vector purification. Clearance of HSV was at least 6.84 log10 with 1% Triton X-100, 4.34 log10 with CIM Q column chromatography, and 2.86 log10 with AVB affinity chromatography. Combined, these three orthogonal methods achieved clearance of at least 14.04 log10 of HSV. The total input quantity of rHSV in a 100-liter production batch is approximately 1.2×10(12) plaque-forming units (pfu), and after purification, the concentration of residual rHSV in the resulting drug substance of approximately 450 ml would be less than 2.42×10(-5) pfu/ml. A rAAV vector produced using this method was used in a clinical trial in which subjects receive up to 100 intramuscular injections of 1.35 ml each, which would contain a maximum of 3.27×10(-3) pfu of HSV. These results support the safety of rAAV vectors produced using our rHSV complementation method.

  6. Recombinant Protein Production in Large-Scale Agitated Bioreactors Using the Baculovirus Expression Vector System.

    PubMed

    Thompson, Christine M; Montes, Johnny; Aucoin, Marc G; Kamen, Amine A

    2016-01-01

    The production of recombinant proteins using the baculovirus expression vector system (BEVS) in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantification, bioreactor preparation and operation, the infection process, final harvesting, and primary separation steps for recovery of the product are presented. Furthermore, methods involved with advanced on-line monitoring and bioreactor control, which have a significant impact on the overall process success, are briefly discussed.

  7. Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination.

    PubMed Central

    Shinagawa, H; Makino, K; Amemura, M; Kimura, S; Iwasaki, H; Nakata, A

    1988-01-01

    The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector. The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins. The proteins encoded by these open reading frames were identified by the maxicell method. The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon. The distal one complemented the ruv mutations. Transcription of the operon was studied both in vivo and in vitro. Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor. A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon. The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins. Images PMID:2842314

  8. Manufacturing of recombinant adeno-associated viral vectors for clinical trials

    PubMed Central

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings. PMID:27014711

  9. Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

    PubMed

    Moore, Lauren; Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

  10. DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

    PubMed Central

    Morrical, Scott W.

    2015-01-01

    The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species. PMID:25646379

  11. DEK is required for homologous recombination repair of DNA breaks.

    PubMed

    Smith, Eric A; Gole, Boris; Willis, Nicholas A; Soria, Rebeca; Starnes, Linda M; Krumpelbeck, Eric F; Jegga, Anil G; Ali, Abdullah M; Guo, Haihong; Meetei, Amom R; Andreassen, Paul R; Kappes, Ferdinand; Vinnedge, Lisa M Privette; Daniel, Jeremy A; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I

    2017-03-20

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

  12. DEK is required for homologous recombination repair of DNA breaks

    PubMed Central

    Smith, Eric A.; Gole, Boris; Willis, Nicholas A.; Soria, Rebeca; Starnes, Linda M.; Krumpelbeck, Eric F.; Jegga, Anil G.; Ali, Abdullah M.; Guo, Haihong; Meetei, Amom R.; Andreassen, Paul R.; Kappes, Ferdinand; Vinnedge, Lisa M. Privette; Daniel, Jeremy A.; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I.

    2017-01-01

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition. PMID:28317934

  13. [Construction of a new bacterial artificial chromosome (BAC) vector for cloning of large DNA fragments and heterologous expression in Streptomyces].

    PubMed

    Huang, Sheng; Li, Na; Zhou, Jun; He, Jing

    2012-01-01

    Many natural product biosynthetic gene clusters are too large to be entirely cloned into one cosmid for heterologous expression. Because bacterial artificial chromosome (BAC) vectors are well known for their capacity of cloning large DNA fragments, we constructed a new BAC vector for cloning and heterologous expression of natural product biosynthesis gene clusters in Streptomyces. The chloramphenicol resistance gene on the original BAC vector pCUGIBAC1 was substituted with a streptomycin resistance gene via lambda RED-mediated PCR-targeting technique. The streptomycin resistance gene was then excised by digestion with NheI and the left gap was filled with the origin of transfer (oriT), the phiC31 integrase gene, the integrating attP site, and an apramycin resistance gene. We achieved the final BAC vector pMSBBACs. To test the newly established vector, pMSBBACs was used to build up a genomic BAC library of Streptomyces U27. The average size of inserts in the library is about 100kb. A 140 kb BAC plasmid as a representative was successfully introduced into heterologous hosts, S. lividans and S. albus, by either conjugation or protoplast transformation. It demonstrated that the BAC plasmids constructed by pMSBBACs could be integrated into chromosomes via site-specific recombination for heterologous expression. The newly constructed pMSBBACs was verified to be a good BAC vector for cloning of large DNA fragments and heterologous expression in Streptomyces.

  14. Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

    PubMed Central

    Lock, Martin; Alvira, Mauricio; Vandenberghe, Luk H.; Samanta, Arabinda; Toelen, Jaan; Debyser, Zeger

    2010-01-01

    Abstract Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV–adenovirus, AAV–herpesvirus, and AAV–baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 × 1014 genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies. PMID:20497038

  15. Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors.

    PubMed

    Lotery, Andrew J; Derksen, Todd A; Russell, Stephen R; Mullins, Robert F; Sauter, Sybille; Affatigato, Louisa M; Stone, Edwin M; Davidson, Beverly L

    2002-04-10

    We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina. We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys. Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes. Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes. rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given. After surgery the animals were clinically evaluated by retinal photography and electroretinography. beta-Galactosidase expression was evaluated, at a final end point, in histological sections. We found photoreceptor and Müller cells to have the greatest transgene expression. Focal inflammatory responses localized to the injection site were seen histologically in all eyes. No difference in transduction efficiency was seen between injections near the macula and more peripheral injections. Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections. We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina. rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye.

  16. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    PubMed

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  17. Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster.

    PubMed Central

    Baker, B S; Boyd, J B; Carpenter, A T; Green, M M; Nguyen, T D; Ripoll, P; Smith, P D

    1976-01-01

    Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing. PMID:825857

  18. Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

    PubMed Central

    2011-01-01

    Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials. PMID:21995317

  19. Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

    PubMed

    Voss, Carsten; Lindau, Dennis; Flaschel, Erwin

    2006-01-01

    The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

  20. A DNA topoisomerase VI-like complex initiates meiotic recombination.

    PubMed

    Vrielynck, Nathalie; Chambon, Aurélie; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; De Muyt, Arnaud; Mézard, Christine; Mayer, Claudine; Grelon, Mathilde

    2016-02-26

    The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure.

  1. Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors.

    PubMed

    Lajoie, C A; Layton, A C; Easter, J P; Menn, F M; Sayler, G S

    1997-10-01

    Degradation of polychlorinated biphenyls (PCBs) in the environment is limited by their aqueous solubility and the degradative competence of indigenous populations. Field application vectors (FAVs) have been developed in which surfactants are used to both increase the solubility of the PCBs and support the growth of surfactant-degrading strains engineered for PCB degradation. Surfactant and PCB degradation by two recombinant strains were investigated. Pseudomonas putida IPL5 utilizes both alkylethoxylate [polyoxyethylene 10 lauryl ether (POL)] and alkylphenolethoxylate [Igepal CO-720 (IGP)] surfactants as growth substrates, but only degrades the ethoxylate moiety. The resulting degradation products from the alkyl- and alkylphenolethoxylate surfactants were 2-(dodecyloxy)ethanol and nonylphenoldiethoxylates, respectively. Ralstonia eutropha B30P4 grows on alkylethoxylate surfactants without the appearance of solvent-extractable degradation products. It also degrades the 2-(dodecyloxy)ethanol produced by strain IPL5 from the alkylethoxylate surfactants. The extent of degradation of the alkylethoxylate surfactant (POL) was greater for strain IPL5 (90%) than for B30P4 (60%) as determined by the cobaltothiocyanate active substances method (CTAS). The recombinant strain B30P4::TnPCB grew on biphenyl. In contrast, the recombinant strain IPL5::TnPCB could not grow on biphenyl, and PCB degradation was inhibited in the presence of biphenyl. The most extensive surfactant and PCB degradation was achieved by the use of both recombinant strains together in the absence of biphenyl. PCB (Aroclor 1242) and surfactant (POL) concentrations were reduced from 25 ppm and 2000 ppm, respectively, to 6.5 ppm and 225 ppm, without the accumulation of surfactant degradation products. Given the inherent complexity of commercial surfactant preparations, the use of recombinant consortia to achieve extensive surfactant and PCB degradation appears to be an environmentally acceptable and effective

  2. Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples

    PubMed Central

    Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre

    2016-01-01

    Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects. PMID:27626642

  3. Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples.

    PubMed

    Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre; Glaizot, Olivier; Fumagalli, Luca

    2016-01-01

    Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects.

  4. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

    PubMed Central

    Kuzminov, Andrei

    1999-01-01

    Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

  5. Would Dissociative Recombination of DNA+ be a Possible Pathway of DNA Damage?

    NASA Astrophysics Data System (ADS)

    Kwon, H. C.; Chen, Z. P.; Strom, R. A.; Andrianarijaona, V. M.

    2015-05-01

    It is known that dissociative recombination (DR) is one of the very efficient processes of destruction of molecular cations into neutral particles. During the past few years, the focus of DR has been expanded from small inorganic molecules to macromolecular cation. We are probing the possibility of the DR of DNA+ after ionization of DNA, for example due to ionizing radiation. Therefore we are investigating the existence of autoionization states within nucleotide bases (Guanine, Adenine, Cytosine, and Thymine). Our results from computational analysis using the modern electronic structure program ORCA will be presented. Authors wish to give special thanks to Pacific Union College Student Senate for their financial support.

  6. Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

    PubMed

    Derse, D; Martarano, L

    1990-01-01

    A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.

  7. New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

    PubMed Central

    Kanno, Alex I.; Goulart, Cibelly; Rofatto, Henrique K.; Oliveira, Sergio C.; Leite, Luciana C. C.

    2016-01-01

    The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. PMID:26850295

  8. New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

    PubMed

    Kanno, Alex I; Goulart, Cibelly; Rofatto, Henrique K; Oliveira, Sergio C; Leite, Luciana C C; McFadden, Johnjoe

    2016-04-01

    The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

  9. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  10. Generation of recombinant bacillus Calmette-Guérin and Mycobacterium smegmatis expressing BfpA and intimin as vaccine vectors against enteropathogenic Escherichia coli.

    PubMed

    Vasconcellos, Halyka Luzorio Franzotti; Scaramuzzi, Karina; Nascimento, Ivan Pereira; Da Costa Ferreira, Jorge M; Abe, Cecilia M; Piazza, Roxane M F; Kipnis, Andre; Dias da Silva, Wilmar

    2012-09-07

    Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in children. EPEC adheres to the intestinal epithelium and causes attaching and effacing (A/E) lesions. Recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express either BfpA or intimin. The entire bfpA gene and a portion of the intimin gene were amplified by PCR from EPEC genomic DNA and inserted into the pMIP12 vector at the BamHI/KpnI sites. The pMIP_bfpA and pMIP_intimin vectors were introduced separately into Smeg and BCG. Recombinant clones were selected based on kanamycin resistance and designated rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin was detected by Immunoblotting using polyclonal anti-BfpA and anti-intimin antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice by assaying the feces and serum for the presence of anti-BfpA and anti-intimin IgA and IgG antibodies. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA, whereas TNF-γ was produced in mice immunized with recombinant intimin. The adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies from mice immunized with recombinant BfpA or intimin but not by antibodies from non-immunized mice. Immunogenic non-infectious vectors containing relevant EPEC virulence genes may be promising vaccine candidates.

  11. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  12. Using RT-prone recombination to promote re-building of complete retroviral vectors from two defective precursors: low efficiency and sequence specificities.

    PubMed

    Bru, Thierry; Galetto, Román; Piver, Eric; Collin, Christine; Negroni, Matteo; Pagès, Jean-Christophe

    2007-06-01

    Retroviral recombination has been suggested as a useful way to modify retroviral vectors. The possibility to combine two multiply deleted retroviral vectors into a novel vector was evaluated. To investigate this possibility we have constructed two defective vectors containing a shared internal ribosome entry site (IRES). The IRES was selected for its complex secondary structure, a feature described to favour retroviral recombination. The IRES was expected to promote a recombination event leading to the formation of a unique, functional retroviral vector. By supporting expression of two transgenes from a single promoter, this sequence was also expected to allow straightforward detection of the recombination event. The present data confirms the achievement of recombination-dependent rescue, albeit at low efficiency. Unexpectedly, a preferential use of the packaging signal (Psi) for recombination was observed, as compared to the IRES. Together these observations mitigate the idea of using this technique for the design of retroviral vectors.

  13. Durable cytotoxic immune responses against gp120 elicited by recombinant SV40 vectors encoding HIV-1 gp120 +/- IL-15.

    PubMed

    McKee, Hayley J; T'sao, Patricia Y; Vera, Maria; Fortes, Puri; Strayer, David S

    2004-08-23

    BACKGROUND: A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency. METHODS: SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120. RESULTS: Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with >/= 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses. CONCLUSIONS: Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV.

  14. Generation, characterization, and docking studies of DNA-hydrolyzing recombinant F(ab) antibodies.

    PubMed

    Zein, Haggag S; El-Sehemy, Ahmed A; Fares, Mohamed O; ElHefnawi, Mahmoud; da Silva, Jaime A Teixeira; Miyatake, Kazutaka

    2011-01-01

    Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs.

  15. Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector against Dengue Virus in Rhesus Monkeys.

    PubMed

    Bischof, Georg F; Magnani, Diogo M; Ricciardi, Michael; Shin, Young C; Domingues, Aline; Bailey, Varian K; Gonzalez-Nieto, Lucas; Rakasz, Eva G; Watkins, David I; Desrosiers, Ronald C

    2017-08-15

    Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 10(5) PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection.IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks

  16. Recombinant Antigens from Phlebotomus perniciosus Saliva as Markers of Canine Exposure to Visceral Leishmaniases Vector

    PubMed Central

    Sumova, Petra; Rohousova, Iva; Jimenez, Maribel; Molina, Ricardo; Volf, Petr

    2014-01-01

    Background Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. Methodology/Principal Findings The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. Conclusions/Significance Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector

  17. Persistence and renaturation efficiency of thermally treated waste recombinant DNA in defined aquatic microcosms.

    PubMed

    Fu, Xiao H; Wang, Lei; Le, Yi Q; Hu, Jia J

    2012-01-01

    To validate the possibility of horizontal gene transfer (HGT) from thermally denatured recombinant DNA discharged into the eco-system, a constructed plasmid was used to investigate the persistence and renaturation efficiency of thermally denatured recombinant DNA in defined aquatic microcosms. The results revealed that there was undecayed recombinant plasmid pMDLKJ material being discharged into the aquatic microcosms even after thermal treatment at either 100°C (using boiling water) or at 120°C (using an autoclave). The plasmid had a relatively long persistence time. At least 10(2) copies μL(-1) of a specific 245 bp fragment of the plasmid could be detected after 12 h and a specific 628 bp fragment could be detected up to 2 h. The thermally denatured recombinant DNA could efficiently renature and recover its functional double stranded structure in aquatic microcosms and the highest concentration of double-stranded DNA (dsDNA) occurred around 1 h after the thermally denatured DNA was added to the system. These results imply that when thermally treated recombinant DNAs are discharged into aquatic environments, they have enough time to renature and possibly transfer to other organisms. In addition, the recombinant DNA added to aquatic microcosms could be absorbed by the seston particles in water, such as mineral, organic and colloids particles with a maximum absorption value of about 5.18 ng L(-1). This absorbed DNA could persist longer in aquatic environments than free recombinant DNA, thus further favoring HGT.

  18. High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

    NASA Astrophysics Data System (ADS)

    Miller, Cynthia K.; Temin, Howard M.

    1983-05-01

    DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

  19. Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

    PubMed

    Chen, Xin Jie

    2013-09-01

    Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

  20. Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

    PubMed Central

    2013-01-01

    SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

  1. Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

    PubMed Central

    Tillman, Robert E.; Wooley, Andrea L.; Hughes, Maureen M.; Wehrly, Tara D.; Swat, Wojciech; Sleckman, Barry P.

    2002-01-01

    Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination. PMID:11828005

  2. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin.

    PubMed

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Miller, Paul E; Sharma, Alok K; Ver Hoeve, James N; Howard, Kellie; Knop, David R; Neuringer, Martha; McGill, Trevor; Stoddard, Jonathan; Chulay, Jeffrey D

    2015-09-01

    Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 10(10) or 4 × 10(11) vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.

  3. Recombination and pseudorecombination driving the evolution of the begomoviruses Tomato severe rugose virus (ToSRV) and Tomato rugose mosaic virus (ToRMV): two recombinant DNA-A components sharing the same DNA-B

    PubMed Central

    2014-01-01

    Background Begomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens since the introduction of a new biotype of the insect vector in the mid-1990’s. Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete sequence of the DNA-B components of ToSRV and ToRMV show an identity of 98.2%. Additionally, the high nucleotide identity (96.2%) between their common regions indicates that these two viruses may share the same DNA-B. Methods Tomato seedlings were biolistically inoculated with ToSRV (DNA-A and DNA-B) and ToRMV (DNA-A and DNA-B) infectious clones in every possible combination of single or mixed infection. Symptom expression was evaluated for up to 35 days post-inoculation (dpi). DNA was extracted at 28 dpi and the presence of each viral genomic component was examined by rolling circle amplification (RCA) followed by digestion, as well as by quantitative, real-time PCR. Sequence comparisons, recombination and phylogenetic analyzes were performed using EMBOSS needle, RDP program and maximum likelihood inference, respectively. Results Symptoms in tomato plants inoculated with the different combinations of ToRMV and ToSRV DNA-A and DNA-B components consisted of a typical mosaic in all combinations. Pseudorecombinants were formed in all possible combinations. When two DNA-A or two DNA-B components were inoculated simultaneously, the ToRMV components were detected preferentially in relation to the ToSRV components. The combination of minor changes in both the Rep protein and the CR may be involved in the preferential replication of ToRMV components. Recombination and phylogenetic analyzes support the exchange of genetic material between ToRMV and ToSRV. Conclusions ToRMV and ToSRV form viable pseudorecombinants in their natural host (Solanum

  4. Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

    PubMed Central

    Li, Lina; Dimitriadis, Emilios K.; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H.; Garcia, Luis; Kotin, Robert M.

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA. PMID:23936358

  5. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    PubMed

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  6. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Edlarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  7. Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration.

    PubMed

    Jelesko, John G; Carter, Kristy; Kinoshita, Yuki; Gruissem, Wilhelm

    2005-09-01

    A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent DeltaRBCS1B: LUC transgene. Sixteen luc(+) (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination.

  8. Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

    PubMed

    Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

    2012-03-01

    Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

  9. Current Good Manufacturing Practice Production of an Oncolytic Recombinant Vesicular Stomatitis Viral Vector for Cancer Treatment

    PubMed Central

    Meseck, M.; Derecho, I.; Lopez, P.; Knoblauch, C.; McMahon, R.; Anderson, J.; Dunphy, N.; Quezada, V.; Khan, R.; Huang, P.; Dang, W.; Luo, M.; Hsu, D.; Woo, S.L.C.; Couture, L.

    2011-01-01

    Abstract Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 109 plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 1010 PFU/ml (total yield, 1 × 1013 PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC. PMID:21083425

  10. Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

    PubMed

    Ausubel, L J; Meseck, M; Derecho, I; Lopez, P; Knoblauch, C; McMahon, R; Anderson, J; Dunphy, N; Quezada, V; Khan, R; Huang, P; Dang, W; Luo, M; Hsu, D; Woo, S L C; Couture, L

    2011-04-01

    Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

  11. Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

    PubMed Central

    Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

    2015-01-01

    Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

  12. Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals

    PubMed Central

    Fuchs, Jonathan D; Bart, Pierre-Alexandre; Frahm, Nicole; Morgan, Cecilia; Gilbert, Peter B; Kochar, Nidhi; DeRosa, Stephen C; Tomaras, Georgia D; Wagner, Theresa M; Baden, Lindsey R; Koblin, Beryl A; Rouphael, Nadine G; Kalams, Spyros A; Keefer, Michael C; Goepfert, Paul A; Sobieszczyk, Magdalena E; Mayer, Kenneth H; Swann, Edith; Liao, Hua-Xin; Haynes, Barton F; Graham, Barney S; McElrath, M Juliana

    2015-01-01

    Background Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. Methods HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. Results All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. Conclusions Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted. PMID:26587311

  13. pGreen-S: a clone vector bearing absence of enhanced green fluorescent protein for screening recombinants.

    PubMed

    Tang, Jinbao; Liang, Shujuan; Zhang, Jinbao; Gao, Zhiqin; Zhang, Suhua

    2009-05-01

    The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5alpha produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.

  14. The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants.

    PubMed

    Peyret, Hadrien; Lomonossoff, George P

    2013-09-01

    The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

  15. Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae.

    PubMed

    Chopra-Dewasthaly, Rohini; Marenda, Marc; Rosengarten, Renate; Jechlinger, Wolfgang; Citti, Christine

    2005-12-01

    Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.

  16. DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.

    PubMed

    Kasai, Kazue; Saeki, Yoshinaga

    2006-06-01

    The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

  17. How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

    PubMed Central

    White, Daniel James; Bryant, David; Gemmell, Neil John

    2013-01-01

    Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

  18. Measurements of DNA-loop formation via Cre-mediated recombination

    PubMed Central

    Shoura, Massa J.; Vetcher, Alexandre A.; Giovan, Stefan M.; Bardai, Farah; Bharadwaj, Anusha; Kesinger, Matthew R.; Levene, Stephen D.

    2012-01-01

    The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo. PMID:22589415

  19. No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

    PubMed Central

    Hagström, Erik; Freyer, Christoph; Battersby, Brendan J.; Stewart, James B.; Larsson, Nils-Göran

    2014-01-01

    Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals. PMID:24163253

  20. A genome-wide map of mitochondrial DNA recombination in yeast.

    PubMed

    Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

    2014-10-01

    In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

  1. A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

    PubMed Central

    Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

    2014-01-01

    In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

  2. Effects of DNA size on transformation and recombination efficiencies in Xylella fastidiosa.

    PubMed

    Kung, Stephanie H; Retchless, Adam C; Kwan, Jessica Y; Almeida, Rodrigo P P

    2013-03-01

    Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.

  3. Distilling Artificial Recombinants from Large Sets of Complete mtDNA Genomes

    PubMed Central

    Kong, Qing-Peng; Salas, Antonio; Sun, Chang; Fuku, Noriyuki; Tanaka, Masashi; Zhong, Li; Wang, Cheng-Ye; Yao, Yong-Gang; Bandelt, Hans-Jürgen

    2008-01-01

    Background Large-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high immanent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable. Methodology/Principal Findings We have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments. Conclusions We have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments – fully concordant with the predictions from our data-analytical approach. PMID:18714389

  4. Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity.

    PubMed

    Ellison, V; Stillman, B

    1998-03-06

    Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells. It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates. This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors. Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex. In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.

  5. Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors.

    PubMed

    Yamashita, Toshiharu; Okura, Masae; Ishii-Osai, Yasue; Hida, Tokimasa

    2016-10-01

    Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP-A to -G, and a variant type (XP-V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP-A, -B, -C, -D, -F or -G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV-C at 5-20 J/m(2) was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP-E and XP-V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co-infection of Ad-XPA with Ad-XPE increased survival rate of XP-E cells after UV-C exposure. When XP-V cell strains, including one derived from a Japanese patient, were infected with Ad-XPV, exposed to UV-B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP-V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP-E and XP-V. © 2016 Japanese Dermatological Association.

  6. [New protein vectors based on an alpha-fetoprotein fragment for targeted DNA delivery into cancer cells].

    PubMed

    Tatarinova, O N; Gorokhovets, N V; Makarov, V A; Posypanova, G A; Serebriakova, M V; Pozmogova, G E

    2010-01-01

    A human alpha-fetoprotein fragment (AFP) modified with oligocationic homologs of nuclear localization signal was used to construct new target cell-selective DNA-carrier proteins. The new recombinant vectors containing C- or N-terminal polynucleotide-binding domains are able to form stable complexes with single- or double-stranded oligonucleotides and plasmid DNA. Using flow cytometry and fluorescent microscopy, it was shown that such nucleoprotein complexes can be selectively internalized in target cells receptors superexpressing AFP receptors. The results obtained are important both for understanding mechanisms of formation of DNA-protein complexes and for studying their interaction with intracellular molecular targets. The new proteins can be used as a tool for the development of highly selective and efficacious gene-selective antitumour drugs.

  7. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  8. [Construction of a recombinant baculovirus transfer vector with two promoters expressing the anti-human CD28 chimeric antibody by using TP-PCR method].

    PubMed

    Zhu, Yan; Chen, Yong-Jing; Qiu, Yu-Hua; Zheng, Feng-Feng; Zhu, Jiang

    2005-09-01

    CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.

  9. Neurovirulence Properties of Recombinant Vesicular Stomatitis Virus Vectors in Non-Human Primates

    PubMed Central

    Johnson, J. Erik; Nasar, Farooq; Coleman, John W.; Price, Roger E.; Javadian, Ali; Draper, Kenneth; Lee, Margaret; Reilly, Patricia A.; Clarke, David K.; Hendry, R. Michael; Udem, Stephen A.

    2007-01-01

    Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for one day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed. PMID:17098273

  10. Exploration of the Dissociative Recombination following DNA ionization to DNA+ due to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Strom, Richard A.; Zimmerly, Andrew T.; Andrianarijaona, Vola M.

    2014-05-01

    It is known that ionizing radiation generates low-energy secondary electrons, which may interact with the surrounding area, including biomolecules, such as triggering DNA single strand and double strand breaks as demonstrated by Sanche and coworkers (Radiat. Res. 157, 227(2002)). The bio-effects of low-energy electrons are currently a topic of high interest. Most of the studies are dedicated to dissociative electron attachments; however, the area is still mostly unexplored and still not well understood. We are computationally investigating the effect of ionizing radiation on DNA, such as its ionization to DNA+. More specifically, we are exploring the possibility of the dissociative recombination of the temporary DNA+ with one of the low-energy secondary electrons, produced by the ionizing radiation, to be another process of DNA strand breaks. Our preliminary results, which are performed with the binaries of ORCA, will be presented. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  11. Production of Recombinant Adeno-Associated Viral Vectors and Use in In Vitro and In Vivo Administration

    PubMed Central

    Gray, Steven J; Choi, Vivian W.; Asokan, Aravind; Haberman, Rebecca A.; McCown, Thomas J.; Samulski, Richard Jude

    2011-01-01

    Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection for many different cell types and its ability to persist and lead to long-term gene expression. This unit describes efficient methods to generate high-titer, research-grade, adenovirus-free recombinant single-stranded and self-complementary adeno-associated virus in various serotypes, along with methods to quantify the viral vectors. Two detailed methods are provided for viral vector delivery into the rodent brain and spinal cord, and for histological detection of transgene expression of GFP. PMID:21971848

  12. [Recombinations occurring in the process of DNA replication in Escherichia coli].

    PubMed

    Sukhodolets, V V

    2006-07-01

    In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.

  13. Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.

    PubMed

    Le Moigne, Vincent; Robreau, Georges; Mahana, Wahib

    2006-01-01

    Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

  14. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

    PubMed

    Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

    2011-12-09

    Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

  15. Scalable Downstream Strategies for Purification of Recombinant Adeno-Associated Virus Vectors in Light of the Properties

    PubMed Central

    Qu, Weihong; Wang, Mingxi; Wu, Yaqing; Xu, Ruian

    2015-01-01

    Recombinant adeno-associated virus (rAAV) vector is one of the promising delivery tools for gene therapy. Currently, hundreds of clinical trials are performed but the major barrier for clinical application is the absence of any ideal large scale production technique to obtain sufficient and highly pure rAAV vector. The large scale production technique includes upstream and downstream processing. The upstream processing is a vector package step and the downstream processing is a vector purification step. For large scale downstream processing, the scientists need to recover rAAV from dozens of liters of cell lysate or medium, and a variety of purification strategies have been developed but not comprehensively compared till now. Consequently, this review will evaluate the scalable downstream purification strategies systematically, especially those based on the physicochemical properties of AAV virus, and attempt to find better scalable downstream strategies for rAAV vectors. PMID:25941887

  16. [Effect of Intron Orientation on the Expression of Transgene Imposed by MAR Expression Vector in Stably Recombinant CHO Cells].

    PubMed

    Li, Qin; Zhao, Chun-peng; Wang, Xiao-yin; Sun, Qiu-li; Wang, Tian-yun

    2016-03-01

    To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector. The MAR of β-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method. The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence. Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.

  17. Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.

    PubMed

    Brantly, Mark L; Spencer, L Terry; Humphries, Margaret; Conlon, Thomas J; Spencer, Carolyn T; Poirier, Amy; Garlington, Wendy; Baker, Dawn; Song, Sihong; Berns, Kenneth I; Muzyczka, Nicholas; Snyder, Richard O; Byrne, Barry J; Flotte, Terence R

    2006-12-01

    A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.

  18. How-to-Do-It: Teaching Recombinant DNA Technology in High School Biology Courses.

    ERIC Educational Resources Information Center

    Dixon, Linda

    1988-01-01

    Reports on the teaching of recombinant DNA technology in high school biology courses. Explains reactions of the public, students, and colleagues to the molecular genetics unit. Indicates equipment, curricular materials, training, workshops, and availability. (RT)

  19. Collaborative Learning in Biology: Debating the Ethics of Recombinant DNA Technology.

    ERIC Educational Resources Information Center

    Anderson, Rodney P.

    1998-01-01

    Discusses applications of recombinant DNA technology and the controversies surrounding that technique. Provides a cooperative learning project idea that involves teams of students investigating and debating these issues. (DDR)

  20. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... attenuated strains of bacteria and viruses that are frequently used in recombinant DNA research. OBA is also... ATCC 6223 (also known as strain B38) Yersinia pestis pgm (-) (lacking the 102 kb pigmentation...

  1. How-to-Do-It: Teaching Recombinant DNA Technology in High School Biology Courses.

    ERIC Educational Resources Information Center

    Dixon, Linda

    1988-01-01

    Reports on the teaching of recombinant DNA technology in high school biology courses. Explains reactions of the public, students, and colleagues to the molecular genetics unit. Indicates equipment, curricular materials, training, workshops, and availability. (RT)

  2. DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

    PubMed

    Hesketh, Emma L; Parker-Manuel, Richard P; Chaban, Yuriy; Satti, Rabab; Coverley, Dawn; Orlova, Elena V; Chong, James P J

    2015-03-20

    ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

  3. Collaborative Learning in Biology: Debating the Ethics of Recombinant DNA Technology.

    ERIC Educational Resources Information Center

    Anderson, Rodney P.

    1998-01-01

    Discusses applications of recombinant DNA technology and the controversies surrounding that technique. Provides a cooperative learning project idea that involves teams of students investigating and debating these issues. (DDR)

  4. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  5. PCR mediated recombination impacts the analysis of hepatitis B Virus covalently closed circular DNA.

    PubMed

    Suspène, Rodolphe; Thiers, Valérie; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2016-12-20

    The replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination. The impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination. PCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.

  6. New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica.

    PubMed

    VanDrisse, C M; Escalante-Semerena, J C

    2016-07-01

    Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications.

  7. Structural Studies of DNA End Detection and Resection in Homologous Recombination

    PubMed Central

    Schiller, Christian Bernd; Seifert, Florian Ulrich; Linke-Winnebeck, Christian; Hopfner, Karl-Peter

    2014-01-01

    DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies. PMID:25081516

  8. Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model.

    PubMed

    Hosseini, Ahmad; Estiri, Hajar; Akhavan Niaki, Haleh; Alizadeh, Akram; Abdolhossein Zadeh, Baharak; Ghaderian, Sayyed Mohammad Hossein; Farjadfar, Akbar; Fallah, Ali

    2017-10-01

    Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×105 rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×107 infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple

  9. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

    PubMed Central

    Hart, Bryan E.; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D.; Lukose, Regy; Souther, Sommer J. R.; Rayasam, Swati D. G.; Saelens, Joseph W.; Chen, Ching-ju; Seay, Sarah A.; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E.; Ng, Tony W.; Tobin, David M.; Porcelli, Steven A.; Larsen, Michelle H.; Schmitz, Joern E.; Haynes, Barton F.; Jacobs, William R.; Lee, Sunhee

    2015-01-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  10. [Inhibition of infectious bursal disease virus replication in chicken embryos by miRNAs delivered by recombinant avian adeno-associated viral vector].

    PubMed

    Shen, Pengpeng; Wang, Yongjuan; Sun, Huaichang; Zhang, Xinyu; Xia, Xiaoli

    2011-02-01

    We studied the inhibition of infectious bursal disease virus (IBDV) replication in chicken embryos by recombinant avian adeno-associated virus (AAAV)-delivered VP1- and VP2-specific microRNAs (miRNAs). We co-transfected AAV-293 cells with the VP1- or VP2 gene-specific miRNA expression vector pAITR-RFPmiVP1 or AITR-RFPmiVP2E, AAAV packaging vector pcDNA-ARC and adenovirus helper vector pHelper, resulting in recombinant virus rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E. We also generated the recombinant viruses rAAAV-RFP (without miRNA expression cassette) and rAAAV-RFPmiVP2con (expressing control miRNA) using the same method as the control purpose. Electron microscopy showed that the recombinant viruses had a typical morphology of AAV. We confirmed the presence of miRNA expression cassette in the recombinant viral genomes by using PCR. Our poly (A)-tailed RT-PCR showed correct expression of the miRNAs in the rAAAV-transduced DF-1 cells. We inoculated the recombinant viruses individually into 8-day-old SPF chicken embryos and then challenged them using Lukert strain IBDV on day 2 after inoculation. Our IBDV titration assay showed that the 50% tissue culture infectious dose (TCID50) of rAAAV-RFP- or rAAAV-RFPmiVP2con-inoculated group was 8.0 log10, whereas the TCID50 of rAAAV-RFPmiVP1-inoculated group decreased to 1.0 and 0.8 log10 on day 3 and 6 after challenge, respectively. Similarly, the TCID50 of rAAAV-RFPmiVP2E-inoculated group decreased to 1.5 and 2.0 log10, respectively. These data suggest that rAAAV can transduce efficiently chicken embryos and the expressed VP1- and VP2-specific miRNAs can inhibit the replication of IBDV efficiently.

  11. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    SciTech Connect

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I.

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  12. Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System

    PubMed Central

    2012-01-01

    Background While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event. Results We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart. Conclusion We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events. PMID:23216697

  13. Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

    PubMed Central

    Ciubotaru, Mihai; Schatz, David G.

    2004-01-01

    V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs. PMID:15367690

  14. Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination.

    PubMed

    Ciubotaru, Mihai; Schatz, David G

    2004-10-01

    V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

  15. Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung.

    PubMed

    Katkin, J P; Husser, R C; Langston, C; Welty, S E

    1997-01-20

    Somatic gene therapy for pulmonary diseases must be accomplished in vivo, requiring the spread of a gene transfer vector across a vast expanse of respiratory epithelium. Surfactant, a naturally occurring protein and lipid mixture used to treat the respiratory distress syndrome of prematurity, disperses rapidly and evenly throughout the lung. We employed exogenous bovine surfactant (Survanta beractant) as a carrier vehicle for pulmonary delivery of a recombinant adenovirus expressing beta-galactosidase (beta-Gal). Rats treated with an adenovirus-beractant mixture demonstrated more uniform lobar distribution of transgene expression than rats treated with the same amount of virus in saline. Tissue homogenates were examined for quantitative beta-Gal expression by reaction with o-nitrophenol beta-n-galactopyranoside (ONPG). The degree of beta-Gal activity was affected by both the volume and type of carrier used to deliver the virus. At low volumes (0.5 ml, 1.3 ml/kg), beractant-treated animals demonstrated significantly greater pulmonary beta-Gal activity than saline-treated animals (p < 0.002) and untreated controls. At high volume (1.2 ml, 4 ml/kg), average beta-Gal activity was similar between groups treated with beractant or saline, but was more variable within the saline treated group. Higher volumes of delivery medium were associated with increased levels of beta-Gal expression regardless of the carrier used. Survanta was well tolerated by the animals and did not affect the duration of transgene expression. Exogenous beractant provides a useful medium for delivering recombinant adenoviruses to the lung when diffuse distribution of transgene expression is desired.

  16. Protective immune response induced by co-immunization with the Trichinella spiralis recombinant Ts87 protein and a Ts87 DNA vaccine.

    PubMed

    Yang, Yaping; Yang, Xiaodi; Gu, Yuan; Wang, Yunyun; Zhao, Xi; Zhu, Xinping

    2013-05-20

    Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge in mice. To determine if a combination of recombinant Ts87 protein and its coding DNA induces a stronger immune response in female C57BL/6 mice were immunized with 100 μg of recombinant Ts87 protein plus its coding DNA cloned in vector pVAX1, or the same amount of recombinant protein or DNA only. Mouse subclass IgG responses showed that both co-immunized and single-immunized mice produced a balanced IgG2a/IgG1 (Th1/Th2) response. T-cell proliferation in co-immunized animals was significantly higher than in single-immunized mice. Cytokine profiling in the co-immunization group showed a significant increase in the levels of IL-2, IL-4, IL-6 and IFN-γ in the splenocytes of mice upon stimulation with the recombinant Ts87 protein; however, the expression of IL-17 was down-regulated. Challenge results showed that mice immunized with the recombinant Ts87 protein and its coding DNA produced reduced the muscle larval burden to a greater extent (43.8%) than the groups immunized with only the protein (39.7%) or the DNA (9.7%). A better Th1/Th2 immune response and consequent protection induced by co-immunization with the recombinant Ts87 protein and its coding DNA may result from an adjuvant effect of DNA and a specific persistent expression of Ts87.

  17. Human insulin genome sequence map, biochemical structure of insulin for recombinant DNA insulin.

    PubMed

    Chakraborty, Chiranjib; Mungantiwar, Ashish A

    2003-08-01

    Insulin is a essential molecule for type I diabetes that is marketed by very few companies. It is the first molecule, which was made by recombinant technology; but the commercialization process is very difficult. Knowledge about biochemical structure of insulin and human insulin genome sequence map is pivotal to large scale manufacturing of recombinant DNA Insulin. This paper reviews human insulin genome sequence map, the amino acid sequence of porcine insulin, crystal structure of porcine insulin, insulin monomer, aggregation surfaces of insulin, conformational variation in the insulin monomer, insulin X-ray structures for recombinant DNA technology in the synthesis of human insulin in Escherichia coli.

  18. Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy.

    PubMed

    Negrete, Alejandro; Esteban, Geoffrey; Kotin, Robert M

    2007-09-01

    A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 x 10(15) particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 x 10(16) particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

  19. Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

    PubMed

    Gowen, Brian B; Ennis, Jane; Russell, Andrew; Sefing, Eric J; Wong, Min-Hui; Turner, Jeffrey

    2011-01-01

    Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

  20. DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation.

    PubMed

    Hah, Young-Sool; Lee, Jung Hwa; Kim, Deok Ryong

    2007-05-31

    V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

  1. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  2. The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis.

    PubMed

    Li, Liangliang; Jean, Martine; Belzile, François

    2006-03-01

    We examined the effects of substrate divergence and DNA mismatch repair (MMR) on recombination in Arabidopsis thaliana. Relative to the frequency observed in plants with a homologous construct (0% divergence), recombination was decreased 4.1-, 9.6-, 11.7- or 20.3-fold, respectively, in lines with constructs containing 0.5%, 2%, 4% or 9% divergence between the recombination substrates. To evaluate the contribution of the MMR system in this decrease, 12 independent reporter lines (two or three lines per reporter construct) were crossed to an AtMSH2 T-DNA insertional mutant. We examined the recombination frequency in progeny homozygous for a reporter T-DNA and homozygous either for the wild type or the mutant allele of AtMSH2. The loss of MMR activity led to a two- to ninefold increase in homeologous recombination and the size of the increase did not seem to correlate with the amount of divergence. Inversely, complementation of the insertional mutant with a wild-type cDNA of AtMSH2 reduced recombination. Our results demonstrate clearly that sequence divergence can dramatically reduce the recombination frequency in plants and that the MMR system plays a part in this decrease.

  3. Joint capsule treatment with enkephalin-encoding HSV-1 recombinant vector reduces inflammatory damage and behavioural sequelae in rat CFA monoarthritis.

    PubMed

    Lu, Ying; McNearney, Terry A; Wilson, Steven P; Yeomans, David C; Westlund, Karin N

    2008-03-01

    This study assessed enkephalin expression induced by intra-articular application of recombinant, enkephalin-encoding herpes virus (HSV-1) and the impact of expression on nociceptive behaviours and synovial lining inflammation in arthritic rats. Replication-conditional HSV-1 recombinant vectors with cDNA encoding preproenkephalin (HSV-ENK), or control transgene beta-galactosidase cDNA (HSV-beta-gal; control) were injected into knee joints with complete Freund's adjuvant (CFA). Joint temperatures, circumferences and nociceptive behaviours were monitored on days 0, 7, 14 and 21 post CFA and vector treatments. Lumbar (L4-6) dorsal root ganglia (DRG) and spinal cords were immunostained for met-enkephalin (met-ENK), beta-gal, HSV-1 proteins and Fos. Joint tissues were immunostained for met-ENK, HSV-1 proteins, and inflammatory mediators Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) and cyclo-oxygenase-2, or stained with haematoxylin and eosin for histopathology. Compared to exuberant synovial hypertrophy and inflammatory cell infiltration seen in arthritic rats treated with CFA only or CFA and HSV-beta-gal, the CFA- and HSV-ENK-treated arthritic rats had: (i) striking preservation of synovial membrane cytoarchitecture with minimal inflammatory cell infiltrates; (ii) significantly improved nociceptive behavioural responses to mechanical and thermal stimuli; (iii) normalized Fos staining in lumbar dorsal horn; and (iv) significantly increased met-ENK staining in ipsilateral synovial tissue, lumbar DRG and spinal cord. The HSV-1 and transgene product expression were confined to ipsilateral lumbar DRG (HSV-1, met-ENK, beta-gal). Only transgene product (met-ENK and beta-gal) was seen in lumbar spinal cord sections. Targeted delivery of enkephalin-encoding HSV-1 vector generated safe, sustained opioid-induced analgesia with protective anti-inflammatory blunting in rat inflammatory arthritis.

  4. Stable integration vector for nutrient broth-based selection of attenuated Listeria monocytogenes strains with recombinant antigen expression.

    PubMed

    Lenz, Laurel L; Huang, William A; Zhou, Chenghui; Li, Zhongxia; Calendar, Richard

    2008-09-01

    Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.

  5. Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens.

    PubMed

    Wang, Jian; Lin, Yahui; Cai, Pengfei; Wang, Heng

    2011-01-01

    The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines.

  6. [Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

    PubMed

    Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

    2014-03-01

    To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

  7. In vitro and in vivo Functional Characterization of Gutless Recombinant SV40-derived CFTR Vectors

    PubMed Central

    Mueller, Christian; Strayer, Marlene S; Sirninger, Jeffery; Braag, Sofia; Branco, Francisco; Louboutin, Jean-Pierre; Flotte, Terence R.; Strayer, David S.

    2009-01-01

    In cystic fibrosis (CF) respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress towards gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF we examined the feasibility of using SV40-derived vectors (rSV40s) which may circumvent some of these obstacles. To accommodate the large CFTR cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We therefore tested whether “gutless” rSV40s could be packaged and were able to express a functional human CFTR cDNA. Results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein which localized to the plasma membrane and restored channel function to CFTR deficient cells. Similarly in vivo experiments delivering rSV40-CFTR to the lungs of Cftr−/− mice resulted in a reduction of the pathology associated with intra-tracheal pseudomona aeruginosa challenge. rSV40-CFTR treated mice had had less weight loss when compared to control treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the BAL. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted. PMID:19890354

  8. Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

    PubMed

    Apitz, Janina; Weihe, Andreas; Pohlheim, Frank; Börner, Thomas

    2013-02-01

    While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

  9. Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

    PubMed

    Lerat, Sylvain; Gulden, Robert H; Hart, Miranda M; Powell, Jeff R; England, Laura S; Pauls, K Peter; Swanton, Clarence J; Klironomos, John N; Trevors, Jack T

    2007-12-12

    The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

  10. Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy

    PubMed Central

    Hackett, Christopher S; Geurts, Aron M; Hackett, Perry B

    2007-01-01

    Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis. PMID:18047689

  11. Self-regulation of recombinant DNA technology in Japan in the 1970s.

    PubMed

    Nagai, Hiroyuki; Nukaga, Yoshio; Saeki, Koji; Akabayashi, Akira

    2009-07-01

    Recombinant DNA technology was developed in the United States in the early 1970s. Leading scientists held an international Asilomar Conference in 1975 to examine the self regulation of recombinant DNA technology, followed by the U.S. National Institutes of Health drafting the Recombinant DNA Research Guidelines in 1976. The result of this conference significantly affected many nations, including Japan. However, there have been few historical studies on the self-regulation of recombinant technologies conducted by scientists and government officials in Japan. The purpose of this paper is to analyze how the Science Council of Japan, the Ministry of Education, Science adn Culture, and the Science and Technology Agency developed self-regulation policies for recombinant DNA technology in Japan in the 1970s. Groups of molecular biologist and geneticists played a key role in establishing guidelines in cooperation with government officials. Our findings suggest that self-regulation policies on recombinant DNA technology have influenced safety management for the life sciences and establishment of institutions for review in Japan.

  12. Designing plasmid vectors.

    PubMed

    Tolmachov, Oleg

    2009-01-01

    Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

  13. A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

    PubMed Central

    Birnboim, H C; Doly, J

    1979-01-01

    A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method. Images PMID:388356

  14. Engineering of bacterial strains and vectors for the production of plasmid DNA.

    PubMed

    Bower, Diana M; Prather, Kristala L J

    2009-04-01

    The demand for plasmid DNA (pDNA) is anticipated to increase significantly as DNA vaccines and non-viral gene therapies enter phase 3 clinical trials and are approved for use. This increased demand, along with renewed interest in pDNA as a therapeutic vector, has motivated research targeting the design of high-yield, cost-effective manufacturing processes. An important aspect of this research is engineering bacterial strains and plasmids that are specifically suited to the production of plasmid biopharmaceuticals. This review will survey recent innovations in strain and vector engineering that aim to improve plasmid stability, enhance product safety, increase yield, and facilitate downstream purification. While these innovations all seek to enhance pDNA production, they can vary in complexity from subtle alterations of the host genome or vector backbone to the investigation of non-traditional host strains for higher pDNA yields.

  15. Immunotherapy for Lewis lung carcinoma utilizing dendritic cells infected with CK19 gene recombinant adenoviral vectors

    PubMed Central

    SUN, Q.F.; ZHAO, X.N.; PENG, C.L.; HAO, Y.T.; ZHAO, Y.P.; JIANG, N.; XUE, H.; GUO, J.Z.; YUN, C.H.; CONG, B.; ZHAO, X.G.

    2015-01-01

    Dendritic cells (DCs) as 'professional' antigen-presenting cells (APCs) initiate and regulate immune responses to various antigens. DC-based vaccines have become a promising modality in cancer immunotherapy. Cytokeratin 19 (CK19) protein is expressed at high levels in lung cancer and many other tumor cells, suggesting CK19 as a potential tumor-specific target for cancer immune therapy. We constructed a recombinant adenoviral vector containing the CK19 gene (rAd-CK19). DCs transfected with rAd-CK19 were used to vaccinate C57BL/6 mice bearing xenografts derived from Lewis lung carcinoma (LLC) cells. The transfected DCs gave rise to potent CK19-specific cytotoxic T lymphocytes (CTLs) capable of lysing LLC cells. Mice immunized with the rAd-CK19-DCs exhibited significantly attenuated tumor growth (including tumor volume and weight) when compared to the tumor growth of mice immunized with rAd-c DCs or DCs during the 24-day observation period (P<0.05). The results revealed that the mice vaccinated with the rAd-CK19-DCs exhibited a potent protective and therapeutic antitumor immunity to LLC cells in the subcutaneous model along with an inhibitive effect on tumor growth compared to the mice vaccinated with the rAd-c DCs or DCs alone. The present study proposes a meaningful mode of action utilizing rAd-CK19 DCs in lung cancer immunotherapy. PMID:26323510

  16. Long-Term Immunological Memory Induced by Recombinant Oral Salmonella Vaccine Vectors

    PubMed Central

    Kohler, James J.; Pathangey, Latha; Hasona, Adnan; Progulske-Fox, Ann; Brown, Thomas A.

    2000-01-01

    We have previously shown that Salmonella enterica serovar Typhimurium expressing the hagB hemagglutinin gene from Porphyromonas gingivalis can induce primary and recall immune responses in serum and secretions in mice; however, the longevity of memory induced by oral Salmonella carriers has not been adequately demonstrated. In this study, we examined the capacity of mice to mount a recall response 52 weeks after primary immunization. Recall responses were seen in serum immunoglobulin G (IgG) and IgA following boosting at week 52, and in most cases, they were equal to or greater than the primary responses. Significant mucosal IgA recall responses in saliva and vaginal wash were also detected following boosting at week 52. In addition, there was a considerable residual response in secretions at week 51, prior to boosting. These results indicate that oral Salmonella vectors can induce long-term memory to recombinant HagB and are particularly effective at inducing long-lasting mucosal responses as well as at inducing the capacity for mucosal recall responses. PMID:10858264

  17. Safety considerations in vector development.

    PubMed

    Kappes, J C; Wu, X

    2001-11-01

    The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5' sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the psi packaging signal joined with an open gag coding region. Analysis of the 3' end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector

  18. Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

    PubMed

    Wang, Gensheng; Young, Sarah P; Bali, Deeksha; Hutt, Julie; Li, Songtao; Benson, Janet; Koeberl, Dwight D

    2014-01-01

    A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease.

  19. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

    PubMed

    Ni, W; Le Guiner, C; Gernoux, G; Penaud-Budloo, M; Moullier, P; Snyder, R O

    2011-07-01

    Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

  20. Novel Infection System of Recombinant BmBDV DNA into BmN Cells of Silkworm, Bombyx mori.

    PubMed

    Guo, Rui; Cao, Guangli; Zhu, Yuexiong; Kumar, Dhiraj; Xue, Renyu; Lu, Yahong; Hu, Xiaolong; Gong, Chengliang

    2016-10-01

    Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

  1. A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair

    PubMed Central

    Jain, Suvi; Sugawara, Neal; Lydeard, John; Vaze, Moreshwar; Tanguy Le Gac, Nicolas; Haber, James E.

    2009-01-01

    A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a “recombination execution checkpoint” (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence. PMID:19204116

  2. Recombinant DNA Paper Model Simulation: The Genetic Engineer.

    ERIC Educational Resources Information Center

    Wagner, Joan

    1998-01-01

    Describes a course for talented high school students that focuses on DNA science and technology. Employs Cold Spring Harbor's DNA Science laboratory manual. Engages students in performing sickle-cell anemia and thalassemia tests in rabbits. (DDR)

  3. Recombinant DNA Paper Model Simulation: The Genetic Engineer.

    ERIC Educational Resources Information Center

    Wagner, Joan

    1998-01-01

    Describes a course for talented high school students that focuses on DNA science and technology. Employs Cold Spring Harbor's DNA Science laboratory manual. Engages students in performing sickle-cell anemia and thalassemia tests in rabbits. (DDR)

  4. Waste recombinant DNA: effectiveness of thermo-treatment to manage potential gene pollution.

    PubMed

    Fu, Xiaohua; Li, Mengnan; Zheng, Guanghong; Le, Yiquan; Wang, Lei

    2009-01-01

    Heating at 100 degrees C for 5-10 min is a common method for treating wastewater containing recombinant DNA in many bio-laboratories in China. In this experiment, plasmid pET-28b was used to investigate decay efficiency of waste recombinant DNA during thermo-treatment. The results showed that the decay half-life of the plasmid was 2.7-4.0 min during the thermo-treatment, and even heating for 30 min the plasmids still retained some transforming activity. Low pH promoted the decay of recombinant DNA, but NaCl, bovine serum albumin and EDTA, which existed in the most wastewater from bio-laboratories, protected DNA from degradation. Thus, the decay half-life of plasmid DNA may be longer than 2.7-4.0 min practically. These results suggest that the effectiveness of heating at 100 degrees C for treating waste recombinant DNA is low and a gene pollution risk remains when those thermo-treated recombinant DNAs are discharged into the environment. Therefore other simple and effective methods should be developed.

  5. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli.

    PubMed Central

    Moreau, P L

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA. PMID:2836358

  6. Switch recombination breakpoints are strictly correlated with DNA recognition motifs for immunoglobulin S gamma 3 DNA-binding proteins

    PubMed Central

    1992-01-01

    The deletion looping out model of switch (S) recombination predicts that the intervening DNA between switch regions will be excised as a circle. Circular excision products of immunoglobulin switch recombination have been recently isolated from lipopolysaccharide (LPS)- stimulated spleen cells. The recombination breakpoints in these large circles were found to fall within switch regions. Since switch recombination is clearly focused on switch regions, we hypothesized that some DNA-binding protein factor might be involved in specifically recognizing and facilitating the alignment of switch regions before recombination. Two DNA-binding proteins that specifically interact with two discrete regions of the S gamma 3 tandem repeat have been identified in crude and partially purified nuclear extracts derived from LPS- and dextran sulfate (DxS)-activated splenic B cells. The first factor has been found indistinguishable from NF-kappa B by mobility shift assays, methylation interference, competition binding studies, and supershift analysis using an antiserum specific for the p50 component. The second appears to be composed of two closely traveling mobilities that do not separate upon partial purification. This second complex is unique and specific for S gamma 3 by methylation interference assays and competition-binding analysis. The sites at which recombination occurs in the S gamma 3 switch region have been analyzed and found to strictly correlate with the binding sites of the S gamma 3 switch binding proteins. PMID:1500850

  7. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

    PubMed

    Wilson, Marenda A; Kwon, YoungHo; Xu, Yuanyuan; Chung, Woo-Hyun; Chi, Peter; Niu, Hengyao; Mayle, Ryan; Chen, Xuefeng; Malkova, Anna; Sung, Patrick; Ira, Grzegorz

    2013-10-17

    During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

  8. A simplified method for purification of recombinant soluble DnaA proteins.

    PubMed

    Zawilak-Pawlik, Anna M; Kois, Agnieszka; Zakrzewska-Czerwinska, Jolanta

    2006-07-01

    An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.

  9. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  10. A simplified mathematical model of directional DNA site-specific recombination by serine integrases

    PubMed Central

    Zhao, Jia; Stark, W. Marshall; Colloms, Sean D.; Ebenhöh, Oliver

    2017-01-01

    Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB. The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both ‘forward’ and ‘reverse’ reactions. In this model, the substrates of the ‘forbidden’ reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein–DNA complexes, ensuring that these ‘forbidden’ reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality. PMID:28077763

  11. Optimization of a new non-viral vector for transfection: Eudragit nanoparticles for the delivery of a DNA plasmid.

    PubMed

    Gargouri, M; Sapin, A; Bouli, S; Becuwe, P; Merlin, J L; Maincent, P

    2009-12-01

    The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the

  12. Rad51 and Rad52 are involved in homologous recombination of replicating herpes simplex virus DNA.

    PubMed

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

  13. Rad51 and Rad52 Are Involved in Homologous Recombination of Replicating Herpes Simplex Virus DNA

    PubMed Central

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells. PMID:25365323

  14. A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

    PubMed

    Uringa, Evert-Jan; Baldeyron, Céline; Odijk, Hanny; Wassenaar, Evelyne; van Cappellen, Wiggert A; Maas, Alex; Hoeijmakers, Jan H J; Baarends, Willy M; Kanaar, Roland; Essers, Jeroen

    2015-01-01

    Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

    PubMed Central

    Thomason, Lynn C.; Costantino, Nina

    2016-01-01

    ABSTRACT Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. PMID:27624131

  16. In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector

    USDA-ARS?s Scientific Manuscript database

    Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE),US3 serine/threonine protein kinase (PK), or b...

  17. Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

    PubMed

    Ren, Z J; Baumann, R G; Black, L W

    1997-08-22

    A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.

  18. A facile method for reversibly linking a recombinant protein to DNA.

    PubMed

    Goodman, Russell P; Erben, Christoph M; Malo, Jonathan; Ho, Wei M; McKee, Mireya L; Kapanidis, Achillefs N; Turberfield, Andrew J

    2009-06-15

    We present a facile method for linking recombinant proteins to DNA. It is based on the nickel-mediated interaction between a hexahistidine tag (His(6)-tag) and DNA functionalized with three nitrilotriacetic acid (NTA) groups. The resulting DNA-protein linkage is site-specific. It can be broken quickly and controllably by the addition of a chelating agent that binds nickel. We have used this new linker to bind proteins to a variety of DNA motifs commonly used in the fabrication of nanostructures by DNA self-assembly.

  19. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  20. The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

    PubMed

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

  1. A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species

    PubMed Central

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun

    2014-01-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

  2. Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

    PubMed

    Lee, Hee-Jung; Hur, Yoon-Ki; Cho, Youn-Dong; Kim, Mi-Gyeong; Lee, Hoon-Taek; Oh, Yu-Kyoung; Kim, Young Bong

    2012-01-01

    Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

  3. Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks.

    PubMed

    Ge, Jinying; Wang, Xijun; Tian, Meijie; Gao, Yuwei; Wen, Zhiyuan; Yu, Guimei; Zhou, Weiwei; Zu, Shulong; Bu, Zhigao

    2015-05-15

    Canine Distemper Virus (CDV) infects many carnivores and cause several high-mortality disease outbreaks. The current CDV live vaccine cannot be safely used in some exotic species, such as mink and ferret. Here, we generated recombinant lentogenic Newcastle disease virus (NDV) LaSota expressing either envelope glycoproyein, heamagglutinine (H) or fusion protein (F), named as rLa-CDVH and rLa-CDVF, respectively. The feasibility of these recombinant NDVs to serve as live virus-vectored CD vaccine was evaluated in minks. rLa-CDVH induced significant neutralization antibodies (NA) to CDV and provided solid protection against virulent CDV challenge. On the contrast, rLa-CDVF induced much lower NA to CDV and fail to protected mink from virulent CDV challenge. Results suggest that recombinant NDV expressing CDV H is safe and efficient candidate vaccine against CDV in mink, and maybe other host species.

  4. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

    SciTech Connect

    Gottlieb, J.; Muzyczka, N.

    1988-06-01

    When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

  5. Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant alphavirus replicon particles.

    PubMed

    Goldberg, Stacie M; Bartido, Shirley M; Gardner, Jason P; Guevara-Patiño, José A; Montgomery, Stephanie C; Perales, Miguel-Angel; Maughan, Maureen F; Dempsey, JoAnn; Donovan, Gerald P; Olson, William C; Houghton, Alan N; Wolchok, Jedd D

    2005-11-15

    Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen. Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection. T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone. Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.

  6. Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells.

    PubMed Central

    Kretschmer, P J; Bowman, A H; Huberman, M H; Sanders-Haigh, L; Killos, L; Anderson, W F

    1981-01-01

    We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes. Images PMID:6273826

  7. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination

    PubMed Central

    Sansam, Christopher L; Pezza, Roberto J

    2015-01-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1. PMID:25953379

  8. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.

    PubMed

    Sansam, Christopher L; Pezza, Roberto J

    2015-07-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1.

  9. Design and construction of improved new vectors for Zymomonas mobilis recombinants.

    PubMed

    Dong, Hong-Wei; Bao, Jie; Ryu, Dewey D Y; Zhong, Jian-Jiang

    2011-07-01

    Zymomonas mobilis is a very important gram-negative bacterium having a potential application to simultaneous co-production of biofuel and other high value-added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis-acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram-negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tc(r) ). The utility of screening marker, lacZα with MCS, was confirmed by the blue-white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a-derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co-production of biofuel and high value added products.

  10. Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).

    PubMed

    Athanasopoulos, T; Graham, I R; Foster, H; Dickson, G

    2004-10-01

    Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene. The size of the gene (2.4 Mb) and mRNA (14 kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of efficient gene therapy approaches. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems (nonpathogenicity and minimal immunogenicity, extensive cell and tissue tropism) but accommodate limited transgene capacity (<5 kb). As a result of these observations, a number of laboratories worldwide have engineered a series of microdystrophin cDNAs based on genotype-phenotype relationship in Duchenne (DMD) and Becker (BMD) dystrophic patients, and transgenic studies in mdx mice. Recent progress in characterization of AAV serotypes from various species has demonstrated that alternative AAV serotypes are far more efficient in transducing muscle than the traditionally used AAV2. This article summarizes the current progress in the field of recombinant adeno-associated viral (rAAV) delivery for DMD, including optimization of recombinant AAV-microdystrophin vector systems/cassettes targeting the skeletal and cardiac musculature.

  11. Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus

    PubMed Central

    Matassov, Demetrius; Marzi, Andrea; Latham, Terri; Xu, Rong; Ota-Setlik, Ayuko; Feldmann, Friederike; Geisbert, Joan B.; Mire, Chad E.; Hamm, Stefan; Nowak, Becky; Egan, Michael A.; Geisbert, Thomas W.; Eldridge, John H.; Feldmann, Heinz; Clarke, David K.

    2015-01-01

    Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7–9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines. PMID:26109675

  12. Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus.

    PubMed

    Matassov, Demetrius; Marzi, Andrea; Latham, Terri; Xu, Rong; Ota-Setlik, Ayuko; Feldmann, Friederike; Geisbert, Joan B; Mire, Chad E; Hamm, Stefan; Nowak, Becky; Egan, Michael A; Geisbert, Thomas W; Eldridge, John H; Feldmann, Heinz; Clarke, David K

    2015-10-01

    Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7-9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines.

  13. Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir).

    PubMed

    Davis, Leroy K

    2014-10-01

    The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb's free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

  14. V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

    PubMed Central

    Boubnov, N V; Wills, Z P; Weaver, D T

    1993-01-01

    Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining. PMID:8413286

  15. DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

    PubMed Central

    Cuomo, C A; Mundy, C L; Oettinger, M A

    1996-01-01

    Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination. PMID:8816481

  16. An inhibitor of DNA recombination blocks memory consolidation, but not reconsolidation, in context fear conditioning.

    PubMed

    Colón-Cesario, Melissa; Wang, Jianpeng; Ramos, Xiomara; García, Hermes G; Dávila, Jorge J; Laguna, Jessenia; Rosado, Claribel; Peña de Ortiz, Sandra

    2006-05-17

    Genomic recombination requires cutting, processing, and rejoining of DNA by endonucleases, polymerases, and ligases, among other factors. We have proposed that DNA recombination mechanisms may contribute to long-term memory (LTM) formation in the brain. Our previous studies with the nucleoside analog 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP), a known inhibitor of DNA ligases and polymerases, showed that this agent blocked consolidation of conditioned taste aversion without interfering with short-term memory (STM). However, because polymerases and ligases are also essential for DNA replication, it remained unclear whether the effects of this drug on consolidation were attributable to interference with DNA recombination or neurogenesis. Here we show, using C57BL/6 mice, that ara-CTP specifically blocks consolidation but not STM of context fear conditioning, a task previously shown not to require neurogenesis. The effects of a single systemic dose of cytosine arabinoside (ara-C) on LTM were evident as early as 6 h after training. In addition, although ara-C impaired LTM, it did not impair general locomotor activity nor induce brain neurotoxicity. Importantly, hippocampal, but not insular cortex, infusions of ara-C also blocked consolidation of context fear conditioning. Separate studies revealed that context fear conditioning training significantly induced nonhomologous DNA end joining activity indicative of DNA ligase-dependent recombination in hippocampal, but not cortex, protein extracts. Finally, unlike inhibition of protein synthesis, systemic ara-C did not block reconsolidation of context fear conditioning. Our results support the idea that DNA recombination is a process specific to consolidation that is not involved in the postreactivation editing of memories.

  17. Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation.

    PubMed

    Prado, F; Piruat, J I; Aguilera, A

    1997-05-15

    The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes. Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination. Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription. We show that the induction of deletions between repeats in hpr1delta cells of Saccharomyces cerevisiae is linked to transcription elongation. Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1delta are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats. Transcription becomes HPR1 dependent when elongating through such regions. Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats. In contrast to previously reported cases of transcription-induced recombination, there was no correlation between high levels of transcripts and high levels of recombination. Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation.

  18. High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

    PubMed

    Fu, Xinping; Wang, Hua; Zhang, Xiaoliu

    2002-06-01

    Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

  19. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Eldarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  20. Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation.

    PubMed Central

    Prado, F; Piruat, J I; Aguilera, A

    1997-01-01

    The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes. Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination. Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription. We show that the induction of deletions between repeats in hpr1delta cells of Saccharomyces cerevisiae is linked to transcription elongation. Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1delta are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats. Transcription becomes HPR1 dependent when elongating through such regions. Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats. In contrast to previously reported cases of transcription-induced recombination, there was no correlation between high levels of transcripts and high levels of recombination. Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation. PMID:9184227

  1. Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

    PubMed

    Ikeda, H; Kobayashi, I

    1977-09-01

    Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.

  2. Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

    PubMed Central

    Ikeda, H; Kobayashi, I

    1977-01-01

    Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination. PMID:333450

  3. [Construction of recombinant lentiviral vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro].

    PubMed

    Shan, Xiu-ying; Liu, Zhao-liang; Wang, Biao; Guo, Guo-xiang; Wang, Mei-shui; Zhuang, Fu-lian; Cai, Chuan-shu; Zhang, Ming-feng; Zhang, Yan-ding

    2011-07-01

    To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells. Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi. Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

  4. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  5. DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

    PubMed Central

    Hyppa, Randy W.; Fowler, Kyle R.; Cipak, Lubos; Gregan, Juraj; Smith, Gerald R.

    2014-01-01

    Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination. PMID:24089141

  6. Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4.

    PubMed

    Branagan, Amy M; Klein, Jenny A; Jordan, Christian S; Morrical, Scott W

    2014-01-31

    The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.

  7. Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

    PubMed

    Gonçalves, Manuel A F V; Holkers, Maarten; van Nierop, Gijsbert P; Wieringa, Roeland; Pau, Maria G; de Vries, Antoine A F

    2008-08-29

    A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further

  8. Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization.

    PubMed

    Liu, Cunxia; Du, Shouwen; Li, Chang; Wang, Yuhang; Wang, Maopeng; Li, Yi; Yin, Ronglan; Li, Xiao; Ren, Dayong; Qin, Yanqing; Ren, Jingqiang; Jin, Ningyi

    2013-06-01

    This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA (pCCMp24) and recombinant attenuated vaccinia virus Tian Tan (rddVTT-CCMp24). Intramuscular immunization was performed on days 0 (prime) and 21 (boost). The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus (HIV)-specific binding antibody levels and cytokine (interleukin-2 and interleukin-4) concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4(+)/CD8(+) cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8(+) T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-γ response, providing a basis for further studies.

  9. Comparison of recombinant α-hemoglobin from Crocodylus siamensis expressed in different cloning vectors and their biological properties.

    PubMed

    Maijaroen, Surachai; Anwised, Preeyanan; Klaynongsruang, Sompong; Daduang, Sakda; Boonmee, Atcha

    2016-02-01

    Hemoglobin (Hb) is an important component in red blood cells of the vertebrate. It is a major respiratory protein with oxygen or carbon dioxide transport function. Hb has been reported to contain bioactive peptides which have antibacterial and antioxidant activities. In this study, the alpha-chain hemoglobin(αHb) gene of Crocodylus siamensis was cloned into the three different expression vectors and expressed in Escherichia coli BL21 (DE3). The recombinant αHb proteins from all constructs could be expressed and purified. The result from UV-visible absorption spectra showed a similar pattern of all recombinant proteins to the oxy-hemoglobin form of intact Hb. The different recombinant αHb could exhibit antioxidant activities. All recombinant proteins could inhibit the growth of Bacillus spp. Especially, most of the recombinant proteins could inhibit the growth of Bacillus amyloliquefaciens TISTR 1045 better than intact one. The result obtained from this study can provide us further information about the possibility using of αHb as a supplementary food. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. The OECD Blue Book on Recombinant DNA Safety Considerations: it's influence on ISBR and EFSA activities.

    PubMed

    Schiemann, Joachim

    2006-01-01

    Biosafety regulatory frameworks are intended to serve as mechanisms for ensuring the safe use of biotechnology products without imposing unacceptable risk to human health or the environment, or unintended constraints to technology transfer. The OECD Blue Book on "Recombinant DNA Safety Considerations", setting out principles and concepts for handling genetically modified organisms safely outside of contained laboratory conditions, was a milestone in the history of biotechnology. The "Recombinant DNA Safety Considerations" definitively became the major resource for the formulation of national regulatory frameworks and international regulations, including the Cartagena Protocol.

  11. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

    PubMed Central

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-01-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application. PMID:26134901

  12. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

    PubMed

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-10-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

  13. DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

    PubMed

    Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R

    2015-10-15

    During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.

  14. Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination.

    PubMed

    Lario, Luciana Daniela; Botta, Pablo; Casati, Paula; Spampinato, Claudia Patricia

    2015-06-01

    The mismatch repair (MMR) system maintains genome integrity by correcting replication-associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, MutSγ, which is a heterodimer: MSH2-MSH7. To further understand the role of MSH7 in vivo, we present data from this protein in Arabidopsis thaliana. First, we generated transgenic plants that express β-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analysed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination.

  15. Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining.

    PubMed

    Oksenych, Valentyn; Kumar, Vipul; Liu, Xiangyu; Guo, Chunguang; Schwer, Bjoern; Zha, Shan; Alt, Frederick W

    2013-02-05

    Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5'-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.

  16. DNA sequences, recombinant DNA molecules and processes for producing bovine growth hormone-like polypeptides in high yield

    SciTech Connect

    Buell, G.N.

    1987-09-15

    This patent describes a process for increasing the yield of a bovine growth hormone-like polypeptide to at least 100 times that of a bovine growth hormone-like polypeptide encoded by a DNA sequence. The process comprises the steps of culturing a host transformed with a recombinant DNA molecule comprising DNA sequence encoding a Met ..lambda.. or ..lambda.. bovine growth hormone-like polypetide operatively linked to an expression control sequence. The ..lambda.. is an amino terminal deletion from the amino acid sequence of mature bovine growth hormone.

  17. [Important points in virus research using recombinant DNA technology].

    PubMed

    Nikaido, Takahiko; Takeuchi, Kaoru

    2007-06-01

    Cartagena Protocol on Biosafety to the Convention on Biological Diversity seeks to protect biological diversity from potential risks posed by living modified organisms (LMOs) resulting from modern biotechnology. This protocol was ratified in Japan after establishing domestic law and regulations for the protocol. In the domestic law, use of LMOs is classified into type 1 use (use without containment measures) and type 2 use (use with containment measures). According to the domestic law, most of experiments using recombinant viruses are required for the approval of the Minister. In this article, we will explain Cartagena Protocol and the Japanese domestic low and indicate an example of application form for the approval of the Minister.

  18. In vivo repopulation of cytoplasmically gene transferred hematopoietic cells by temperature-sensitive mutant of recombinant Sendai viral vector.

    PubMed

    Yoshida, Kumi; Yonemitsu, Yoshikazu; Tanaka, Sakura; Yoshida, Shuro; Shibata, Satoko; Kondo, Haruhiko; Okano, Shinji; Ishikawa, Fumihiko; Akashi, Koichi; Inoue, Makoto; Hasegawa, Mamoru; Sueishi, Katsuo

    2007-09-28

    Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.

  19. Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

    PubMed Central

    Souza, Ana Paula; Andrade, Bruno Bezerril; Aquino, Dorlene; Entringer, Petter; Miranda, José Carlos; Alcantara, Ruan; Ruiz, Daniel; Soto, Manuel; Teixeira, Clarissa R.; Valenzuela, Jesus G.; de Oliveira, Camila Indiani; Brodskyn, Cláudia Ida; Barral-Netto, Manoel; Barral, Aldina

    2010-01-01

    Background Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure. Methodology/principal findings ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples. Conclusion Our results show the possibility of substituting Lu. longipalpis SGS for two

  20. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    PubMed

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  1. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks

    PubMed Central

    Bacman, Sandra R.; Williams, Sion L.; Moraes, Carlos T.

    2009-01-01

    To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. ScaI cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR. Curiously, we found that some ScaI sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of ScaI cleavage sites, confirming the recombinogenic character of DSBs in mtDNA. A region adjacent to the D-loop was preferentially involved in recombination of all molecules. Sequencing through NZB and BALB haplotype markers in recombined molecules enabled us to show that in addition to intra-molecular mtDNA recombination, rare inter-molecular mtDNA recombination events can also occur. This study underscores the role of DSBs in the generation of mtDNA rearrangements and supports the existence of recombination hotspots. PMID:19435881

  2. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    SciTech Connect

    Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

    1984-01-01

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig.

  3. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis

    PubMed Central

    Northall, Sarah J.; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L.

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming “Displacement loops” (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  4. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

    PubMed

    Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

    2016-04-05

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

  5. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

    PubMed Central

    Nolden, T.; Pfaff, F.; Nemitz, S.; Freuling, C. M.; Höper, D.; Müller, T.; Finke, Stefan

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. PMID:27046474

  6. Recombinant cccDNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model.

    PubMed

    Li, Gaiyun; Zhu, Yuanfei; Shao, Dianhui; Chang, Hao; Zhang, Xiaoming; Zhou, Dongming; Gao, Yueqiu; Lan, Ke; Deng, Qiang

    2017-07-27

    Covalently closed circular (ccc) DNA of hepatitis B virus (HBV) is critical for viral persistence in vivo. We recently reported a technique involving recombinant (r) cccDNA of HBV by site-specific DNA recombination. Using hydrodynamic injection, rcccDNA induces a temporarily prolonged HBV anigenemia in immunocompetent mice, similar to acute resolving HBV infection. In this study, we simulated the pathophysiological impact of chronic hepatitis by which to reproduce (r)cccDNA persistence in mouse models. We showed that rcccDNA achieved long-lasting persistence in the presence of a compromised immune response or when transcriptional activity was repressed. To closely mimic chronic hepatitis, we used a replication-defective recombinant adenoviral (Ad) vector to deliver rcccDNA to the liver, which led to prominent HBV persistence throughout the experiment duration (>62 weeks) in Alb-Cre transgenic mice. A sustained necroinflammatory response and fibrosis were identified in mouse livers, with dysplastic lesions commonly seen during the late stage of viral persistence, analogous to the progressive pathology of clinical chronic hepatitis. (r)cccDNA was intrinsically stable in vivo, enabling long-term persistence in the context of chronic hepatitis. Viral persistence, in turn, may promote progression of chronic liver disease. Our study also presented a valuable surrogate model of HBV cccDNA persistence in mice that could greatly advance our understanding of the pathogenesis of chronic hepatitis B. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

  7. Distribution of Exchanges upon Homologous Recombination of Exogenous DNA in Xenopus Laevis Oocytes

    PubMed Central

    Carroll, D.; Lehman, C. W.; Jeong-Yu, S.; Dohrmann, P.; Dawson, R. J.; Trautman, J. K.

    1994-01-01

    Homologous recombination between DNA molecules injected into Xenopus oocyte nuclei was investigated by examining the recovery of information from differentially marked parental sequences. The injected recombination substrate was a linear DNA with terminal direct repeats of 1246 bp; one repeat differed from the other by eight single base-pair substitutions, distributed throughout the region of homology, each of which created or destroyed a restriction enzyme site. Recombination products were recovered and analyzed for their content of the diagnostic sites, either directly by Southern blot-hybridization or after cloning in bacteria. The majority (76%) of the cloned products appeared to be the result of simple exchanges-i.e., there was one sharp transition from sequences derived from one parent to sequences derived from the other. These simple exchanges were concentrated near the ends of the homologous interval and, thus, near the sites of the original molecular ends. Placing marked sites on only one side of the homologous overlap showed that marker recovery was governed largely by the positions of the molecular ends and not by the markers themselves. When a terminal nonhomology was present at one end of the substrate, the yield of recombinants was sharply decreased, but the pattern of exchanges was not affected, suggesting that products from end-blocked substrates arise by the same recombination pathway. Because of considerable evidence supporting a nonconservative, resection-annealing mechanism for recombination in oocytes, we interpret the distribution of exchanges as resulting from long-patch repair of extensive heteroduplex intermediates. PMID:7828826

  8. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

    PubMed

    Thomason, Lynn C; Costantino, Nina; Court, Donald L

    2016-09-13

    Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single

  9. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure.

    PubMed

    Yang, Haijuan; Jeffrey, Philip D; Miller, Julie; Kinnucan, Elspeth; Sun, Yutong; Thoma, Nicolas H; Zheng, Ning; Chen, Phang-Lang; Lee, Wen-Hwa; Pavletich, Nikola P

    2002-09-13

    Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.

  10. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    PubMed

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  11. Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

    NASA Technical Reports Server (NTRS)

    Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

    1997-01-01

    Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

  12. Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

    NASA Technical Reports Server (NTRS)

    Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

    1997-01-01

    Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

  13. A Citizen Court in the Recombinant DNA debate.

    ERIC Educational Resources Information Center

    Krinsky, Sheldon

    1978-01-01

    Harvard scientists were planning DNA experiments which required special facilities. A citizen panel was formed to look into the adequacy of federal safety guidelines for the community. Describes the review process and discusses the concept of a citizen court to resolve such technical controversies. (GA)

  14. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging

    PubMed Central

    Singh, Dharmendra Kumar; Ahn, Byungchan

    2009-01-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging. PMID:19083132

  15. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    PubMed Central

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.

    2016-01-01

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  16. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging.

    PubMed

    Singh, Dharmendra Kumar; Ahn, Byungchan; Bohr, Vilhelm A

    2009-06-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging.

  17. Impact of DNA vector topology on non-viral gene therapeutic safety and efficacy.

    PubMed

    Sum, Chi H; Wettig, Shawn; Slavcev, Roderick A

    2014-01-01

    Gene therapy continues to grow as an emerging treatment strategy toward numerous diseases. However, such prospects are hindered by the use of viral vectors prompting significant safety concerns along with limitations concerning repeat administrations, size of delivered gene construct, scale-up, high production costs, contamination during production, and lack of desired tissue selectivity. Non-viral gene delivery demonstrates the potential to address the abovementioned limitations, but itself generally suffers from low efficacy. Continuing efforts have been made to develop innovative delivery systems, synthetic gene carriers, and DNA vectors in a concerted attempt to enhance gene delivery suitable for clinical applications. In this review, we focus on the advances in the design of novel DNA vectors catered to enhance transfection and transgene expression and their influences on the efficacy and safety of existing and emerging delivery systems and synthetic vectors for non viral gene delivery.

  18. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  19. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia.

    PubMed

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem ar