Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells

DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E. coli gpt recombinant gene. The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation. The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants). irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions. However, irs2 rejoined such lesions as correctly as the V79 cells. The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.     

Stability and expression of bacterial genes in replicating geminivirus vectors in plants.

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.     

The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia

Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.     

Chromosomal integration pattern of a helper-dependent minimal adenovirus vector with a selectable marker inserted into a 27.4-kilobase genomic stuffer

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a neo(r) gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable neo(r) transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes.     

Differential expression of two linked selection genes (HSVI-tk and Eco.gpt) in transformed teratocarcinoma and in L cells

Upon transfection of (TK-)F9 teratocarcinoma stem-cells and (TK-)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI-tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV-gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk-regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low.     

Artificial and engineered chromosomes: developments and prospects for gene therapy

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals.     

用慢病毒载体制备转基因动物的研究进展

慢病毒是逆转录病毒的一种 ,具有逆转录病毒的基本结构 ,但也有不同于逆转录病毒的组份和特性 ,作为基因治疗载体发展起来 ,最近已用于转基因动物制备。慢病毒像其它逆转录病毒一样 ,其基因组经逆转录后能整合在宿主DNA上 ;由于病毒载体经改构后 ,不在宿主细胞增殖 ,不会导致寄主细胞的死亡 ,被它感染的或转化的动物细胞能够连续传代 ;这种载体的最大优势是能感染静止细胞和不产生嵌合体动物。详细介绍了慢病毒载体构建原理、近年慢病毒载体在转基因动物制备方法和应用研究的新进展。     

The enhanced transfer of drug-resistant genes in NIH-3T3 cells transformed by the EJras oncogene

The spontaneous transfer of drug resistance genes has been shown to take place between cultured mammalian NIH-3T3 cells and occurs with a hierarchy of transfer efficiencies, transformed cells being more efficient than non-transformed cells. This experiment was accomplished by co-cultivating two NIH-3T3 sublines, each transfected by standard plasmid methods with a different drug resistance gene, subjecting the mixed population to double selection by adding both drugs to the mixed cell culture, and isolating single cells which were resistant to both drugs. The genes used were the neo gene and gpt gene which conferred resistance to the drugs G418 and mycophenolic acid, respectively. DNA analysis confirmed the presence of both resistance genes in the cells which were resistant to both drugs. The mechanism of this gene transfer was by cell fusion rather than by chromosomal DNA uptake. The efficiency of gene transfer, as indicated by the number of double-resistant colonies standardized by number of cells cultured, was much higher between two sublines of cells transformed by the EJras oncogene than between one transformed and one non-transformed subline, which in turn was higher than between two non-transformed sublines. The higher efficiency of gene transfer between the transformed cells also occurred when these cells were injected into nude mice, thus demonstrating that the same process occurred in vivo. It would appear that drug resistance genes may be transferred spontaneously in cultured mammalian cells by cell fusion, and that transformed cells have a higher efficiency of gene transfer compared to non-transformed cells.     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.     

DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors.

None of the vector systems currently available for gene therapy applications have been shown to be capable of both efficient gene transfer into nondividing cells and long-term expression through stable integration into host cell DNA. While integrating vectors based on adeno-associated virus are capable of mediating gene transfer into nondividing cells, this process is 200-fold less efficient than transduction of dividing cells. We demonstrate that the transduction efficiency of adeno-associated virus vectors can be increased by treatment with DNA-damaging agents. Nondividing cells are especially responsive, with increases in transduction efficiency of up to 750-fold. This finding has the potential to facilitate gene therapy applications requiring gene transfer to nondividing cells.     

Replication-defective vectors of reticuloendotheliosis virus transduce exogenous genes into somatic stem cells of the unincubated chicken embryo.

Replication-defective vectors derived from reticuloendotheliosis virus were used to transduce exogenous genes into early somatic stem cells of the chicken embryo. One of these vectors transduced and expressed the chicken growth hormone coding sequence. The helper cell line, C3, was used to generate stocks of vector containing about 10(4) transducing units per ml. Injection of 5- to 20-microliters volumes of vector directly beneath the blastoderm of unincubated chicken embryos led to infection of somatic stem cells. Infected embryos and adults contained unrearranged integrated proviral DNAs. Embryos expressed the transduced chicken growth hormone gene and contained high levels of serum growth hormone. Blood, brain, muscle, testis, and semen contained from individuals injected as embryos contained vector DNA. Replication-defective vectors of the reticuloendotheliosis virus transduced exogenous genes into chicken embryonic stem cells in vivo.     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system

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 investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).