Development of Inducible Gene Targeting in Mice

为了建立基于CRE/LOX策略的可诱导基因打靶系统，利用塔诺昔芬可诱导的Cre重组酶，我们将Cre-ERT2基因导入到小鼠的增殖细胞核抗原基因座中，从而诱导小鼠体细胞中的基因活性发生改变。Cre-ERT2的cDNA在内源性ATG起始密码子后的框架中被引入，然后是一个mc1neo盒，其两侧是loxP序列。或者，使用IRES-Cre-ERT2构建另一个载体。两种载体均含有来自外显子1下游的1.3kb的Apai-Xbal片段和来自内源性的增殖细胞核抗原基因ATG起始密码子上游的7.2kb的片段。经Southern印迹分析，34个克隆为同源重组体，从而建立了PCNA-Cre-ERT2敲入ES细胞系。目前，我们正在通过将ES细胞注射到C57BL/6囊胚中来产生嵌合体。

To establish an inducible gene targeting system, which relies on a Cre/lox-based strategy making use of a tarnoxifen-inducible Cre recombinase, we have introduced a Cre-ERT2 cDNA into the mouse PCNA locus, allowing to induce modification of gene activity in somatic cells of mouse.A genomic fragment corresponding to intron 1 of mouse PCNA gene was amplified from mouse genomic DNA by PCR and was used as a probe to obtain mouse PCNA genomic clones from a mouse J1 ES cell genomic library. cDNA of Cre-ERT2 was introduced in frame after the endogenous ATG start coden in exon 1 of PCNA gene and was followed by a mc1neo cassette flanked by loxP sequences. Alternatively, the IRES-Cre-ERT2 was used to construct another vector. Both constructs contained a 1.3kb ApaI-Xbal fragment from the region downstream of exon1 and an 7.2kb fragment from the region upstream of the endogenous ATG start coden of PCNA gene.Targeting vectors were electroporated into the J1 ES cell line and 295 colonies resistant to G418 were isolated. Thirty-four out of 64 clones tested were identified as homologous recombinat by Southern blot analysis.We, therefore, have established the PCNA-Cre-ERT2 knock-in ES cell lines. We are at present producing chimeras by ES cell injection into C57bl/6 blastocysts.