Cre重组酶调控的OVA-HBsAg转基因乙肝模型小鼠的制备及鉴定

Establishment and identification of OVA-HBs Ag transgenic mice regulated by Cre recombinase

目的制备Cre重组酶调控的卵清蛋白(OVA)-HBs Ag转基因小鼠,为乙肝的防治提供更好的动物模型。方法采用原核显微注射方法将线性化的携带OVA-HBs Ag基因并带有Lox P位点的质粒注入C57BL/6J×DBA小鼠受精卵的雄原核内,制备受Cre重组酶调控表达的OVA-HBs Ag转基因小鼠。将F1代OVA-HBs Ag阳性母鼠与本室饲育的Alb-Cre转基因阳性公鼠进行杂交,获得子代小鼠,观察Cre对OVA-HBs Ag转基因小鼠HBs Ag的诱导表达情况。采用PCR、ELISA和免疫组化方法检测HBs Ag基因、Cre基因在转基因小鼠体内的整合及表达情况。结果共注射受精卵491枚,成活337枚,成活率68.6%。产下F0代小鼠29只,其中PCR阳性4只,外源基因整合率13.8%。目前已传至F4代,F1-F4代PCR阳性率分别为27.5%、32.0%、22.9%、25.0%,ELISA法未检测到血清中HBs Ag表达。将F1代OVA-HBs Ag阳性母鼠与Alb-Cre阳性公鼠杂交,获得16只子代小鼠,PCR检测Cre基因和HBs Ag基因双阳性的子代小鼠有6只,其中2只小鼠血清HBs Ag检测为阳性,诱导表达阳性率为33.3%。结论成功制备出OVA-HBs A转基因小鼠,且可稳定传代,Cre重组酶可以诱导小鼠体内HBs Ag的表达。

Objective To breed OVA-HBs Ag transgenic mice regulated by Cre recombinase in order to provide a better animal model for the study of HBV prevention and therapy. Methods The OVA-HBs Ag transgenic mice were generated by microinjection of OVA-HBs Ag gene with Lox P sites into the pronucleus of C57BL/6J×DBA zygotes. Pups of F1 OVA-HBs Ag female mice cross-fertilized with Alb-Cre male mice were assayed for the expression of HBs Ag induced by Cre recombinase. PCR, ELISA and immunohistochemical methods were used to detect the integration and expression of HBs Ag gene and Cre gene in the transgenic mice. Results 491 fertilized eggs were injected and a total of 337 survived. The survival rate of injection was 68.6%. 29 F0 pups were produced with 4 PCR-positive mice, and the positive rate was 13.8%. Up to now, F4 pups were obtained. The positive rates from F1 to F4 were 27.5%, 32.0%, 22.9% and 25.0%, respectively. No HBs Ag-positive mice were found among these pups. Furthermore, among 16 pups of F1 OVA-HBs Ag female mice crossed with Alb-Cre male mice, 6 were positive for both HBs Ag and Cre as detected by PCR. There were 2 pups showed positive HBs Ag by ELISA assay. The expression rate of HBs Ag after Cre recombinase induction was 33.3%. Conclusion OVA-HBs Ag transgenic mice has been reproduced successfully, and it can be stably passaged. Cre recombinase can induce the expression of HBs Ag in vivo.