BAC介导的含97kb人β类珠蛋白基因簇转基因鼠模型的建立  

Establishment of BAC Mediated Transgenic Mice Containing 97 kb β-globin Gene Cluster

作　　者：沈伟[1] 黄粤[1] 唐毅[1] 刘德培[1] 刘光[1] 吴敏[1] 梁植权[1] 

机构地区：[1]中国医学科学院

出　　处：《中国医学科学院学报》2003年第2期117-121,共5页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金重大项目(39893320)资助

摘　　要：目的 利用RecA蛋白介导的同源重组的方法,修饰含全长人β珠蛋白基因簇及一段IL-11受体α链基因的(?)C(细菌人工染色体)克隆,建立只包括全长人β珠蛋白基因簇的转基因鼠模型。方法 分别在待删除片段上、下游用PCR合成500 bp左右的两段同源序列,共同克隆人构建载体pBV的HindⅢ和XbaⅠ位点之间,然后以SalⅠ酶切后将此1 kb的插入片段再克隆入温度敏感型穿梭载体pSV-RecA中,转化含B5-BAC DNA的感受态大肠杆菌DH10B,经氯霉素正筛选和镰孢菌酸(FA)负筛选,获得发生两次同源重组后只含修饰后的BACDNA而穿梭载体已丢失的菌株。经脉冲场凝胶电泳鉴定后,纯化修饰的BAC DNA,经显微注射受精卵制备转基因鼠,分析其中的人β珠蛋白基因簇整合和表达状况。结果 利用RecA蛋白介导的同源重组的方法,获得了IL-11受体α链基因被定位删除的含完整人β珠蛋白基因簇的BAC克隆(命名为βD-BAC),建立了3株独立的转基因鼠株系。结论 新的BAC介导的转基因鼠中的人β珠蛋白基因簇显示正确的表达模式和水平。Objective To delete IL-11 receptor a chain gene from the Bacterial Artificial Chromosome (BAC) chimerie DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole β-globin gene cluster. Methods Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor a chain gene respectively were cloned into the Hind Ⅲ and Xba Ⅰ sites of pBV vector, then the 1 kb A +B fragment was recovered from the building vector and inserted into the SaiⅠ site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor a chain gene was finally deleted from the BAC DNA through chloramphenieol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice. Results By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor a chain gene from the BAC DNA containing the complete β-globin Gene Cluster and established 3 respective transgenic mice lines. Conclusion Human β-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.

关 键 词：β类珠蛋白 基因簇 转基因鼠模型 RecA蛋白 细菌人工染色体 

分 类 号：Q78[生物学—分子生物学]