Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.     

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.     

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.     

Targeted integration of DNA using mutant lox sites in embryonic stem cells.

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albertet al. (1995)Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox Por LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.     

Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.     

Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells

A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.     

通用型基因打靶载体的构建及其功能鉴定

为了构建适合大多数基因座位点打靶的通用型基因打靶载体及打靶成功后去除正选择标记基因,以克隆载体pGEM-3Z为骨架,插入了一个正选择标记基因新霉素磷酸转移酶基因(neo).两个相同的负选择标记基因单纯疱疹病毒胸苷激酶基因HSV-tk1和HSV-tk2,并在neo的两侧各添加了一个方向相同的LoxP(10cus of crossing-over(X)in P1)序列及两个不同的多克隆位点序列,从而构建了载体pA2T.插入的两个不同的多克隆位点序列中,neo和HSV-tk1之间的多克隆位点序列有8个稀少的酶切位点、neo和HSV-tk2之间的多克隆位点序列有5个稀少的酶切位点,neo、HSV-tk1和HSV-tk2有各自独立的转录单元.脂质体法转染山羊成纤维细胞,用遗传霉素(G418)和丙氧鸟苷(GAC)进行正负筛选,验证了正负选择标记基因的生物活性,证明通用型基因打靶载体pA2T构建成功.栽体pA2T转化组成性表达Cre重组醇(Cyclization recombination protein)的大肠杆菌BM25.8,检测到LoxP序列的生物活性,结果表明pA2T中的正选基因可以被Cre重组酶去除.因此,本研究所构建的通用型基因打靶载体pA2T,根据不同的基因座设计同源臂后,插入到MCS中可直接用于不同基因座位点的打靶,并能够在打靶成功后用Cre重组酶去除基因组中插入的neo基因,为用基因打靶的方法制作转基因动物提供了便利.     

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.     

Restrictase free generation of targeting vectors for disruption of complex mouse genes

Molecular cloning of targeting vectors (TgVs) is a prerequisite procedure for gene disruption in embryonic stem cells. In cases where target genes display complex features (e.g., gene overlap, alternative exon usage), TgVs must mediate deletions with very high precision to prevent unwanted effects. This is often difficult to achieve by procedures using restriction endonucleases and DNA ligases. Therefore, to prepare TgVs for inactivation of two complex genes of immunological interest: PTPRF and NWC, we employed an alternative method, which involves engineering bacterial artificial chromosomes (BACs) by inducible, plasmid encoded "Red/ET recombinase" expression system. Here, we report rapid and efficient construction of PTPRF and NWC TgVs without using restriction endonucleases.