基于凝血因子IX基因剔除小鼠建立血友病乙转基因动物模型

Establishment of A Hemophilia B Transgenic Mouse Model on the Basis of Coagulation Factor IX Gene Knock-out Mouse

为在凝血因子IX基因剔除小鼠基础上建立基因组中整合有含特定点突变的人凝血因子IX基因表达载体的转基因小鼠家系 ,为血友病乙的研究提供更接近临床实际的动物模型。利用体外定点突变技术 ,构建含有特定点突变的人凝血因子IX基因表达载体 ,该载体包括由人凝血因子IX编码区及第一内含子构成的人凝血因子IX基因(hFIXml)、4个拷贝的MCK增强子 (MCKe)、鸡 β -肌动蛋白启动子 (bA)及PolyA ,命名为pMe4bAIXml质粒。将其线性化后 ,用显微注射法注射入 817只凝血因子IX基因剔除小鼠受精卵雄原核 ,再将它们分别回输 4 5只假孕受体母鼠的输卵管中 ,共产仔 6 9只 ,存活 6 3只。采用PCR与基因组Southern杂交筛选法鉴定小鼠 ,证实 6只小鼠基因组中整合有含特定点突变的pMe4bAIXml质粒 ,并对 1只小鼠的PCR产物进行测序 。

This study aimed to introduce a site specific point mutation into the human coagulation factor IX gene expressing vectors (pMe4bAIXml plasmid)for microinjection and to obtain transgenic mouse containing copies of a stably integrated pMe4bAIXml plasmid on the basis of coagulation factor IX gene knock out mouse model as an more efficient animal model of hemophilia b. The site specific point mutation was introduced into pMe4bAIXml plasmid which consists of human coagulation factor IX gene including the entire coding sequence and a shortened first intron, four copies of the mouse MCK enhancer, chicken β actin promotor and poly A signal sequence. The vector was linearized and injected into 817 fertilized eggs of mice in which coagulation factor IX gene has been knocked out. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant females, from which 63 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Six mice were found to carry copies of the intact pMe4bAIXml plasmid containing a point mutation and used as founders to establish transgenic mouse lineages.