Development of Inducible Gene Targeting in Mice

小鼠诱导基因靶向的开发

• 批准号: 13680906
• 负责人: TAKANO Hiroshi
• 金额: $ 0.51万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680906/
• 关键词: mouse; inducible gene targeting; Cre recombinase; tamoxifen; mutated estrogen receptr; PCNA; Cre組み換え酵素

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.

为了建立一个可诱导的基因靶向系统，该系统依靠基于CRE/LOX的策略来利用塔诺氧蛋白诱导的CRE重物组织酶，我们将CRE-ERT2 cDNA引入了小鼠PCNA基因座中，从而允许通过小鼠的体外pcna pcne andne pcna诱导基因的含量。从小鼠J1 ES基因组文库中获得小鼠PCNA基因组克隆的探针。在PCNA基因的外显子1中的内源性ATG起始代码子之后，将CRE-ERT2的cDNA引入框架中，然后是LOXP序列两侧的MC1Neo盒。或者，使用IRES-CRE-ERT2来构建另一个向量。这两种构建体都包含来自Exon1下游区域的1.3kb Apai-Xbal片段，以及来自PCNA基因的内源性ATG启动代码子上游区域的7.2kb片段。将靶向载体电孔量隔离到J1 ES细胞系中，并隔离了295个Colonies colonies colonies colonies colesies colesies coloconies colesies coloconies selyains saligation。在64个测试的克隆中，有34个通过Southern印迹分析鉴定为同源重组。目前，我们正在通过ES细胞注射到C57BL/6胚泡中产生嵌合体。