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.

为了建立一种依赖于利用他诺昔芬诱导型Cre重组酶的基于Cre/lox的策略的诱导型基因靶向系统，我们将Cre-ERT 2 cDNA引入小鼠PCNA基因座，通过PCR从小鼠基因组DNA中扩增对应于小鼠PCNA基因内含子1的基因组片段，并将其用作探针，以获得小鼠PCNA基因，来自小鼠J1 ES细胞基因组文库的PCNA基因组克隆将Cre-ERT 2的cDNA在PCNA基因外显子1的内源性ATG起始科登后的读码框中引入，随后是侧翼为loxP序列的mc 1 neo盒。或者，IRES-Cre-ERT 2用于构建另一种载体。两种载体均含有PCNA基因外显子1下游1.3kb的ApaI-XbaI片段和内源性ATG起始科登上游7.2kb的ApaI-XbaI片段。经Southern印迹分析，在64个克隆中有34个克隆为同源重组，建立了PCNA-Cre-ERT 2基因敲入ES细胞系。我们目前通过将ES细胞注射到C57 bl/6囊胚中来生产嵌合体。