Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.     

影响动物细胞同源重组发生与基因打靶效率的分子机制

真核细胞的基因打靶是基因结构与功能研究的一种非常有价值的技术,也是可应用于基因治疗的具有潜力的工具。有2个限制因素束缚真核细胞基因打靶的发展,即同源重组(HR)率非常低而随机整合率非常高。通过特定基因的过表达或表达干涉,使一些参与DNA重组的蛋白表达水平瞬间改变,可能会增加HR率,降低随机整合率。本文列举了一些与HR相关的候选基因,详细介绍了其中的Rad52上位簇基因,还讨论了打靶载体的设计与修饰、DNA转染方法的有效性等。     

Gene Knockout of the Intracellular Amylase Gene by Homologous Recombination in Streptococcus bovis

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular α-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15–20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. Received: 26 August 1996 / Accepted: 18 December 1996     

Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway

Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two proteins involved in nonhomologous end joining (NHEJ), DNAPKcs and ligase IV, exhibit differential effects on AAV site-specific integration. DNAPKcs is not required; its presence increases the frequency of junction formation indicative of site-specific integration, but seems to reduce the ratio of site-specific integration to random integration (i.e., the latter is even more enhanced). In contrast, site-specific integration is significantly reduced relative to random integration in cells deficient in ligase IV expression. Furthermore, we show that single-stranded AAV vectors are better substrates for site-specific integration than are self-complementary AAV vectors; the absence of DNAPKcs did not affect the targeted integration of these double-stranded AAV vectors. Together, these data suggest that NHEJ proteins participate in site-specific integration, and indicate a role for the single-stranded form of AAV DNA in targeted integration.Adeno-associated virus (AAV) is a ubiquitous human virus (∼80% of adults are seropositive [4]); like other nuclear DNA viruses, it causes persistent infections. Productive infection by AAV requires coinfection with either an adenovirus or herpesvirus (1, 21). In cell culture, an AAV latent infection is readily established by infection with a high multiplicity of infection (MOI) (>250 infectious particles/cell) in the absence of a helper virus coinfection, and such latent infections have been reported to persist for over 100 passages (3). Because of the stability of latent infections, it has been possible to clone latently infected cells and to determine the molecular characteristics of the persistent viral genome. More than 65 to 90% of such clones have been determined to have the AAV genome integrated at a specific site on chromosome 19q13.4 (16, 26). The degree of specificity of the integration site is unique among human viruses. Integration is not specific at the nucleotide level, but a specific target sequence has been determined, which includes a binding site for the AAV Rep protein (RBS) and a so-called terminal resolution site (TRS) which is cleaved in one strand by the Rep protein during AAV DNA replication (19). In addition to the target site, which has been termed AAVS1, site-specific integration has been demonstrated to require the AAV rep protein (either Rep 68 or 78) and a sequence in the inverted terminal repeat homologous to AAVS1 (31, 33). A third Rep binding site (RBS) is found in the promoter at map position 5, which has been reported to greatly enhance site-specific integration (24).Although there have been numerous studies of the mechanisms involved in site-specific integration, many aspects remain to be elucidated. In particular, when during the cell cycle site-specific integration occurs is unknown, and the cellular proteins which are involved have not been identified. Such integration might occur as the consequence of nonhomologous end joining (NHEJ) or homologous recombination (HR). Although the former seems to be a more likely pathway, because integration is into a region of very limited homology, the fact that there is some homology between the minimal essential target sequence and the AAV inverted terminal repeat (ITR) suggests that homologous recombination cannot be arbitrarily dismissed as a possibility. Another unknown feature of site-specific integration is the molecular state of the AAV substrate, i.e., whether the substrate is single or double stranded. Transfection of AAV-containing plasmids does lead to site-specific integration (24); thus, a circular form of duplex AAV DNA can serve as the initial substrate, although it has been more challenging to detect significant levels of such integration after transfection with linear duplex AAV DNA (J. Dyall and K. I. Berns, unpublished data).There have been several studies of the fate of AAV vectors used in gene therapy. The studies have been performed primarily in mice and on occasion in vitro. AAV vectors lack the Rep gene and thus do not preferentially integrate into AAVS1 (25). However, random integration at a low frequency, less than 10⁻⁷ (5), does take place and is of concern because of the potential for insertional mutagenesis and the attendant possibility of oncogenesis (20, 22). Such studies have indicated that NHEJ is involved and that different tissues seem to have variable capacities to support integration (14). Other studies have studied recombination between the ITRs at the ends of AAV DNA. Of particular note have been such studies of AAV vectors containing self-complementary genomes (8). These genomes contain complementary sequences separated by an ITR sequence, which itself is a hairpin sequence. Consequently, these genomes can hairpin on themselves to form a duplex structure which contains three hairpinned ITRs, the one in the middle and the two on the ends. The hairpinned ends are substrates for recombination mediated by NHEJ.If NHEJ were involved in AAV integration, either random or site specific, it would seem likely that an animal deficient in a major component of NHEJ would show less evidence of vector integration. The DNA-dependent protein kinase catalytic subunit is an integral component of NHEJ and is the locus of the underlying genetic defect in immunodeficient SCID mice. Song et al. (30) compared AAV integration both in vitro using cell extracts from wild-type C57BL/6 and SCID mice (derived from C57BL/6) and in vivo. The in vitro assay had been developed as an assay for site-specific integration. Interestingly, the presence of a DNA-dependent protein kinase catalytic subunit (DNAPKcs) in the wild-type extract appeared to inhibit AAV integration. The in vivo results were comparable. Hepatectomies were performed on animals 2 weeks after administration of AAV vectors carrying green fluorescent protein (GFP) as the transgene. Approximately 75% of the liver was removed, and regeneration was allowed to take place. In the SCID mice, a significant percentage (greater than 40%) of hepatocytes still expressed GFP, while GFP fluorescence was lower (less than 10%) in the regenerated wild-type livers. These results were interpreted to mean that either integration was more frequent in SCID mice and/or more stable; thus, DNAPKcs was inhibitory to persistent vector integration (which allowed transgene expression).In this paper, we report experiments designed to assess the effects of mutants which lead to defects in NHEJ on site-specific integration by AAV. We have also compared frequency and stability of site-specific integration relative to those of integration at other sites for both AAV containing single-stranded genomes and those with self-complementary genomes.     

p53 Gene Targeting by Homologous Recombination in Fish ES Cells

Background

Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).

Methodology and Principal Findings

Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.

Conclusions

Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.     

利用细菌人工染色体同源重组对小鼠基因进行条件型敲除

目的:探索通过细菌人工染色体(BAC)同源重组系统构建条件基因敲除载体的高效率方法,提高条件基因敲除小鼠(Flox小鼠)的构建效率。方法:利用作者自己构建的噬菌体重组酶系统,通过BAC同源重组进行条件型基因敲除载体构建工作。首先通过亚克隆构建了一系列载体含有同源臂的靶向质粒,线性化后,打靶片段经电穿孔法转入大肠杆菌内,与相应的BAC同源重组,再经过三步同源重组和一步位点特异性重组,构建小鼠条件型基因敲除载体。结果:高效率构建了小鼠基因的最终条件基因敲除载体。结论:通过BAC同源重组高效构建条件基因敲除载体,为条件基因敲除载体的构建提供了全新思路,并为FLox小鼠的建立,及相应基因在发育、生理、致病机制等方面的功能研究奠定了基础。     

The Population and Evolutionary Dynamics of Homologous Gene Recombination in Bacteria

In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination—broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT)—plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.     

Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

Recombination between Homologous Chromosomes Induced by Unrepaired UV-Generated DNA Damage Requires Mus81p and Is Suppressed by Mms2p

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.     

BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.     

Intrachromosomal Recombination in Saccharomyces cerevisiae : Reciprocal Exchange in an Inverted Repeat and Associated Gene Conversion

Intrachromosomal gene conversion has not shown a strong association with reciprocal exchanges. However, reciprocal exchanges do occur between intrachromosomal repeats. To understand the relationship between reciprocal exchange and gene conversion in repeated sequences the recombination behavior of an inverted repeat was studied. We have found that in one orientation a single copy of the kanr gene of the bacterial transposon Tn903 flanked by part of the inverted repeats IS903 does not give G418 resistance in Saccharomyces cerevisiae. A reciprocal exchange in the IS903 repeats inverts the kanr gene, which then gives G418 resistance in a single copy. Using this as a selection for intrachromosomal reciprocal exchange we have introduced multiple restriction site heterologies into the IS903 repeats and examined the crossover products for associated gene conversions. Approximately 50% of crossovers, both in mitosis and meiosis, were associated with a gene conversion. This suggests that these crossovers result from an intermediate that gives a gene conversion in 50% of the events, that is, both reciprocal exchange and gene conversion between repeated sequences have a common origin. The data are most consistent with a heteroduplex mismatch repair mechanism.     

The Efficiency of Homologous Recombination and Non-Homologous End Joining Systems in Repairing Double-Strand Breaks during Cell Cycle Progression

This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5–24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.     

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.