Targeting frequency for deletion vectors in embryonic stem cells.

We analyzed the gene targeting frequencies and recombination products generated by a series of replacement deletion vectors which target the hprt (hypoxanthine phosphoribosyltransferase) locus in mouse embryonic stem cells. We found that the targeting frequency of a 19.2-kb deletion was comparable to that of a 3-kb deletion or a conventional replacement event in which a 1.7-kb fragment was inserted into the locus. We also observed different integration patterns for these deletion vectors. A result of this finding is that a wide range of genomic deletions in embryonic stem cells is feasible.     

High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.     

Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10¹²–10¹³ mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.     

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.     

Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors.

Replacement vectors with genomic DNA originating from different mouse strains were used to introduce site-specific mutations into the creatine kinase M (CKM) gene of mouse embryonic stem (ES) cells. Here we demonstrate that in mouse strain 129-derived ES cells, the gene is at least 25-fold more efficiently targeted with an isogenic, 129-derived vector (129-pRV8.3) than with a nonisogenic, BALB/c-specific vector (BALB/c-pRV8.3). The two targeting constructs were identical except for allelic differences which were typed by partial sequencing. These included base pair mismatches (2%) and a polymorphic [GTC]-repeat length variation. Both in separate transfections as well as in cotransfections with mixed vectors, homologous disruption of the CKM gene resulted uniquely from the 129-isogenic DNA. Our data confirm earlier observations on requirements for homologous recombination in pro- and eukaryotic systems and indicate that targeting of the CKM locus is highly sensitive to small sequence differences between cognate segments in the endogenous and incoming DNA.     

The effects of terminal heterologies on gene targeting by insertion vectors in embryonic stem cells.

We have examined the effects of placing nonhomologous DNA on the ends of an insertion-type gene targeting vector. The presence of terminal heterologies was found to be compatible with insertion targeting, and the terminal heterologies were efficiently removed. Terminal heterologies reduced the frequency of gene targeting to variable extents. The degree of inhibition of targeting was dependent on the length and the position of the heterology: 2.1kb heterologous sequences were more inhibitory than shorter regions of heterology, and heterology placed on the end of the long (4.8kb) arm of homology was more inhibitory than heterology positioned on the end of the short (0.8kb) arm. When heterology was placed on both arms of the targeting vector the targeting efficiencies were similar to or higher than when heterology was present on the long arm only. These results suggest that terminal sequences are removed simultaneously from both ends of targeting vectors. The removal of terminal sequences probably occurs by exonucleolytic degradation of both strands at each end, and removal of at least one of the strands is intimately coupled with the process of homologous recombination. These findings have implications for the design of gene targeting vectors.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development.

Mouse embryonic stem cell lines offer an attractive route for introducing rare genetic alternations into the gene pool since the cells can be pre-screened in culture and the mutations then transmitted into the germline through chimera production. Two applications of this technique that seem ideally suited for a genetic analysis of development are enhancer and gene trap screens for loci expressed during gastrulation and production of targeted mutations using homologous recombination. These approaches should greatly increase the number of mouse developmental mutants available and help to elucidate the genetic hierarchy controlling embryogenesis.     

Engineering targeted viral vectors for gene therapy

To achieve therapeutic success, transfer vehicles for gene therapy must be capable of transducing target cells while avoiding impact on non-target cells. Despite the high transduction efficiency of viral vectors, their tropism frequently does not match the therapeutic need. In the past, this lack of appropriate targeting allowed only partial exploitation of the great potential of gene therapy. Substantial progress in modifying viral vectors using diverse techniques now allows targeting to many cell types in vitro. Although important challenges remain for in vivo applications, the first clinical trials with targeted vectors have already begun to take place.     

Bi-allelic gene targeting in mouse embryonic stem cells

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.     

Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.     

Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells.

The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter.     

介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎干细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成半乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从半乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这些方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,半乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。     

Towards targeted mutagenesis and gene replacement in plants

Advances in the development of biotechnological tools for plant gene disruption and repair have lagged behind the rapid progress made in whole-genome sequencing of many model and crop plant species. Plant DNA-repair machinery predominantly uses non-homologous end-joining (NHEJ), making the homologous recombination (HR)-based methods, which have proved fruitful for gene targeting in non-plant systems, unsuitable for use in plant systems. Two recent reports describe successful targeted mutagenesis and gene targeting in Arabidopsis by either harnessing the plant NHEJ machinery using site-specific induction of double-strand breaks (DSBs), or by activation of a HR pathway through overexpression of a yeast DNA recombination gene in transgenic plants. These reports provide a foundation from which new technologies for site-specific genome alterations in plant species can be developed.     

Cotransformation and gene targeting in mouse embryonic stem cells.

We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.