基于Cre/Loxp系统Tyro3、Axl受体酪氨酸激酶转基因小鼠的建立

摘要

Objective Use escherichia coli β- galactase gene LacZ as report gene,established Tyro3, Axl receptor tyrosine kinases transgenic mouse containing loxp sites, mating with different tissue specific Crc mice to obtain space-time expressions of transgenic mice, make a foundation for research Tyro3 and Axl in each organization functions.Methods Constructing Tyro3, Axl receptor tyrosine kinase plasmid containing loxp sites, sequencing correctly,through microscopic injection technology to insert the Geo-STOP-msAxl and Geo-STOP-msTyro3 gene containing CAG promoter upstream into BDF1 fertilized eggs,Transplant to ICR receptor mouse, Stay after breeding,using double primers PCR to appraisal F0 and Fn generation mouse genotype, using RT - PCR, X-gal staining to observe LacZ genes' express in mice organization, using WB to detect the protein expression in transgenic mice and wild type.Result Adopt double primer PCR to appraisal new born mice, get modified positive transgenic mouse,use the RT-PCR conformed the exist of LacZ gene;the x-gal staining, observed blue precipitation in the brain,testicular and the thymus of Geo-STOP-msTyro3, observed blue precipitation in brain, heart, ovarian, kidney of GeoSTOP-msAxl,indirectly illustrate the expression part of aim gene;For identification of double primers positive GeoSTOP-msTyro3, Geo-STOP-msAxl transgenic mice and wild type mice, we use the WB to validate the Axl and Tyro3 gene expressing no difference, and explain the exist of STOP sequence, so the Axl and Tyro3 ' s expression is restrained.Conclusions Successful got Geo-STOP-msAxl and Geo-STOP-msTyro3 tyrosine receptor transgenicmouse,%目的 采用大肠杆菌β-半乳糖酶基因LacZ作为报告基因,建立Tyro3、Axl受体酪氨酸激酶含有loxp位点的转基因小鼠.与不同组织特异性Cre小鼠杂交,获得时空性表达Tyro3、Axl的转基因小鼠,为研究其在各个组织中的功能奠定基础.方法 构建含有loxp位点的Tyro3、Axl受体酪氨酸激酶质粒,测序正确后,通过显微注射法将插入CAG启动子下游的Geo-STOP-msAxl及Geo-STOP-msTyro3基因的转基因载体注射人BDF1供体鼠受精卵,移植受精卵至ICR受体鼠,待产仔后,通过双引物PCR鉴定F0以及Fn代小鼠的基因型,RT-PCR,X-gal染色观察小鼠组织中LacZ基因,运用WB(Western Blot)检测转基因小鼠和野生型小鼠体内目的蛋白表达的差异.结果 获得了含有目的基因的转基因阳性小鼠;通过RT-PCR对阳性小鼠证明LacZ基因的存在;对存在LacZ基因的小鼠进行Xgal染色,在Geo-STOP-msTyro3的脑、睾丸、胸腺中观察到蓝色沉淀,在Geo-STOP-msAxl的脑、心、肾中观察到蓝色沉淀,间接说明目的基因能表达的组织和器官;对双阳性的Geo-STOP-msTyro3、Geo-STOP-msAxl转基因小鼠和野生型小鼠.WB检验证实Tyro3、Axl基因的表达没有差异,说明在加入stop序列之后,Tyro3基因确实存在,而且表达受到抑制.结论 成功建立Geo-STOP-msAxl及Geo-STOP-msTyr03酪氨酸受体转基因小鼠.