延胡索酰乙酰乙酸水解酶基因敲除大鼠胚胎干细胞株的建立

Establishment of fumarylacetoacetate hydrolase gene knockout rat embryonic stem cell strains

背景:小鼠肝损伤模型已被广泛应用,大鼠肝损伤模型能在发病机制,肝移植环境等方面更好地模拟人,建立大鼠延胡索酰乙酰乙酸水解酶(fuarylacetoacetat hydrolase,Fah)基因敲除的胚胎干细胞株是大鼠肝损伤模型建立的重点和难点。目的:建立Fah基因缺失的大鼠胚胎干细胞株。方法:根据Genbank检索出的大鼠Fah基因序列构建带有正负双向筛选系统的Fah基因敲除载体pKO-Fah,并进行大鼠胚胎干细胞制备与培养,通过电穿孔转染将线性化质粒转入大鼠胚胎干细胞中,用嘌呤霉素和增强型绿色荧光进行克隆的筛选,再对筛选出来的阳性克隆进行PCR验证。结果与结论:电穿孔转染后观察到绿荧光胚胎干细胞克隆,经过3轮嘌呤霉素药物筛选后约有90%的胚胎干细胞克隆表达绿荧光,成功挑取到2个稳定表达绿荧光的胚胎干细胞克隆,重组胚胎干细胞-Fah,通过PCR鉴定为正确同源重组的阳性克隆。提示大鼠胚胎干细胞稳定建系后,能通过传统的同源重组方法来建立基因敲除胚胎干细胞,并为后期基因敲除动物的建立奠定基础。

BACKGROUND： Mouse liver injury model has been widely used. The pathological mechanism of liver injury and the environment of liver transplantation in rats can be used for simulation in humans. Establishing fuarylacetoacetat hydrolase gene knockout embryonic stem cell strains is the key to establish a rat model of liver injury.OBJECTIVE： To establish fuarylacetoacetat hydrolase gene knockout rat embryonic stem cell strains. METHODS： We first constructed the targeting vector in vitro with positive-negative selective cassette, then transfered the linearized vector into embryonic stem cells through electroporation. We used embryonic stem cell culture medium containing 0.5 μg/mL puromycin to select puromycin resistant clones 36 hours after electroporation, and then we performed PCR to confirm targeted embryonic stem cell clones.RESULTS AND CONCLUSION： After electroporation, about 90% of embryonic stem cell clones expressed green fluorescence. Two embryonic stem cell clones expressing green fluorescence were selected and confirmed by PCR. The fuarylacetoacetat hydrolase gene deficient rat embryonic stem cell strains were successfully established. This provides evidence for later establishing gene knockout animal models.