基于Cre-LoxP系统的条件性定点敲入人源hRas基因小鼠的建立

目的 构建基于Cre- LoxP系统的条件性定点敲入人源hRas基因(c-Ha-ras)的小鼠，以获得hRas基因在特定组织器官条件性表达的小鼠模型，用于药物的临床前致癌性安全评价及相关机制研究。方法 首先构建hRas打靶载体，电击法转染入小鼠胚胎干细胞(ES细胞)，用正负法筛选阳性ES细胞，通过PCR、 Southern鉴定后，将正确重组hRas基因的ES细胞导入C57BL/6 J小鼠囊胚，移入同步发育的受体鼠子宫，妊娠足月出生的嵌合体小鼠与C57BL/6 J小鼠交配获得杂合子hRasfl/+小鼠。再将杂合子hRasfl/+小鼠间交配获得纯合子小鼠hRasfl/fi，然后与全身组织细胞表达Cre重组酶的Tg (EIIa-cre)小鼠进行交配，获得全身细胞表达hRas基因的hRas-EIIa-cre小鼠，并通过荧光定量PCR方法检测不同日龄胚胎期仔鼠的hRas基因表达水平。结果 成功构建了基于Cre- LoxP系统的人源hRas基因条件性定点敲入小鼠模型的载体，筛选得到的一个正确克隆，电转ES细胞后进行Southern blot鉴定，经过初筛和复筛，共获得12个阳性克隆，挑选A11号克隆ES细胞进行囊胚注射，移植了48枚胚胎，出生9只小鼠，其中6只为嵌合鼠，将嵌合率>50%的雄鼠与野生型C57BL /6 J雌鼠进行交配，出生21只后代，其中鉴定有4只hRasfl/+小鼠；hRasfl/+小鼠间交配出生29只小鼠，其中有14只纯合子hRasfl/fi小鼠；hRasfl/fl小鼠与Tg (EIIa-cre)工具鼠交配6次，未有仔鼠出生；跟踪不同发育时期胚胎中hRas基因的表达发现，E10.5～15.5 d胚胎中检测到hRas基因表达。结论 成功建立运用Cre- LoxP系统建立了带有hRas基因敲入的纯合子小鼠hRasfl/fl，未能得到全身细胞高效表达人源hRas基因的hRas-EIIa-cre小鼠，但为进一步利用hRasfl/fl小鼠模型建立其他组织特异性的条件性基因敲入小鼠模型做好技术储备。

Generation of Human hRas Geneconditional Knockin Mouse Using The Cre-LoxP Strategy

Objective Generation of conditional human Rasgene (c-Ha-ras) knock in mouse model by Cre-LoxP strategy for preclinical drug carcinogenicity safety evaluationand related mechanism studies．Method Targeting vector of hRas conditional knock in was constructed and then transfected into embryonic stem (ES) cells by electroporation. ES cells were screened by positive and negative selection, and targeted cells were confirmed by PCR and Southern blot. The targeted ES cells were microinjected into the blastula of C57BL/6 J mice. Chimerical mice were generated by transplanting the blastula into the host mice.hRasfl/+ heterozygous mice were obtained by crossing chimerical founder mice with C57BL/6 J mice. hRasfl/flhomozygote mice were obtained by crossing between hRasfl/+ heterozygous mice. Conventional expression of hRas was induced by crossing between EIIa-cretransgenic mice and hRasfl/fl mice. Quantitative polymerase chain reaction (QPCR) was performed to detect hRas gene expression level in mice embryo at variety of developmental stages. Result Targeting vector based on Cre-LoxP system was constructedand was confirmedby PCR and restriction enzyme digestion.A total of 12 positive ES clones were confirmed by Southern blot by both of 5′-end probeflank and 3′-end flank probe. Clone A11 was chosen and subjected to microscopic blastocyst injection. Forty eight embryos were transplanted into host mice and 9 offspring were obtained, among which six pups were chimeric. Four hRasfl / + heterozygous mice were generated by crossing chimerical mice (chimeric rate>50%) with C57BL/6 J mice.hRasfl / + were intercrossed and 29 offspring were obtained. Fourteen out of 29 offspring were homozygous. In order to obtain hRas- EIIa- cre mice, hRasfl/fl were crossed with EIIa-cre mice for 6 rounds of mating. However, no alive pups were obtained.Further analysis showed that hRas gene expressed during E10.5—15.5 day in the embryo by quantitative polymerase chain reaction (QPCR). Conclusion hRasfl/flhomozygote mice was obtained successfully though we failed to obtain the hRas- EIIa- cre mice due to embryonic lethal. However, establishment of hRasfl/fl mice model provided foundation for generating conditional knockout mice model