慢病毒载体法制备荧光素酶转基因小鼠

目的基于慢病毒介导的转基因方法制备荧光素酶(Luc)转基因小鼠。方法制备携带Luc基因的慢病毒,将其注入小鼠单细胞受精卵卵周隙以感染受精卵,然后将胚胎移植进假孕母鼠体内以获得仔鼠,应用小动物活体成像仪及PCR等在蛋白和DNA水平上筛选和鉴定Luc转基因小鼠。结果移植慢病毒隙感染后的成活胚胎63枚。将其移植至3只假孕母鼠,其中2只怀孕,共生仔鼠11只;利用小动物活体成像仪检测Luc表达,在蛋白水平证实11只F0代中,3只(命名为S1、S2、S3)表达Luc;DNA水平检测证实,3只Luc阳性小鼠的基因组中整合有外源转基因Luc。此外,Luc转基因首建鼠基因组中整合的Luc转基因可稳定遗传至下一代,并能正常表达。Luc转基因小鼠主要脏器如睾丸、肾脏、胃、肠、肺、脑、胸腺、肝脏和心脏等均可见Luc信号,但不同脏器间Luc强度有差异。结论成功制备Luc报告基因转基因小鼠。

Generation of the luciferase transgenic mice by lentivirus-mediated gene delivery

Objective To generate luciferase (Luc) transgenic mice by lentivirus-mediated delivery of foreign genes into zygotes. Methods Luc transgenic mice were generated by the subzonal injection of lentivirus harboring Luc gene into single-cell fertilized eggs of mice to infect fertilized eggs,and subsequently embryos infected by lentivirus were transplanted into the pseudopregnant mice to attain F0 mice,followed by screening Luc transgenic mice from potential founders via Luc assay by using the Xenogen IVIS Lumina Imaging System at birth, and subsequently confirm the results of Luc assay by PCR-based genotyping. Results 63 virus-injected eggs were transplanted into 3 pseudopregnant mice to attain 11 potential transgenic founders. Three Luc-positive mice (marked by S1, S2 and S3) were found via Luc assay 2 days after birth. PCR-based genotyping indicated that Luc gene actually integrated into the genome of three Luc-positive F0 mice, as confirmed by the results of Luc assay. Furthermore, Luc transgene could be transmitted from founders to subsequent generation (F1 progeny). Luc could be detected in different adult organs including testis, kidney, stomach, intestine, lung, brain, thymus, liver and heart. Conclusion Luc transgenic mice were successfully generated.