潮霉素和新霉素抗性基因转基因小鼠的建立及其表达研究  

Generation of Transgenic Mice for Hygromycin and Neomycin Resistance Genes and Studies on Transgene Expression

作　　者：党素英[1] 麻孙恺 孙霞 严兰珍 王铸钢[1] 

机构地区：[1]上海南方模式生物研究中心

出　　处：《生物工程学报》2005年第1期159-162,共4页Chinese Journal of Biotechnology

基　　金：国家杰出青年科学基金项目 (No 3 992 5 0 2 3 ) ;国家高技术研究发展计划项目 (No 2 0 0 1AA2 160 81) ;上海市科技发展基金项目 (No 99JC14 0 2 9;99XD14 0 0 5 )。~~

摘　　要：建立潮霉素 (hygromycin ,hyg)和新霉素 (neomycin ,neo)抗性基因转基因小鼠系 ,以获得两种抗性基因同时表达的小鼠胚胎成纤维细胞 (MEFs) ,为条件性基因剔除或小鼠胚胎干细胞克隆的筛选创造条件。将pTK hygR pA ,PGK neoR pA两个转录单元分别克隆至pBluescript载体中获得hygR neoR 双抗性基因表达质粒 ,以KpnⅠ和XbaⅠ双酶切 ,回收 4 2 4 5bp串联基因片段 ,经显微注射获得HygR neoR 转基因小鼠。PCR及Southernblot鉴定转基因小鼠基因型 ;RT PCR检测hygR、neoR 基因在转基因阳性小鼠组织及MEFs中的表达。经显微注射共获得 7只转基因阳性小鼠 (Founders,G0代 ) ,经交配繁育 ,建立了 6个转基因小鼠系。RT PCR检测其中 5个转基因阳性小鼠系F1代杂合子成年雌性小鼠肝脏、卵巢组织中hygR 、neoR 基因的表达 ,发现hn30 ,hn33,hn6 6和hn6 7系阳性小鼠的 1种或 2种组织中有hygR 、neoR 的表达 ;RT PCR检测发现hyg和neo抗性基因在从hn30和hn6 6系分离培养的MEFs中表达。通过显微注射 ,建立了表达hyg neo抗性基因的转基因小鼠系 ,转基因表达检测发现两个转基因小鼠系的胚胎成纤维细胞中表达双抗性基因。To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock out and screening of drug resistant ES cell clones. To construct Hyg R neo R expression vector, pTK hyg R pA and PGK neo R pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn Ⅰ and Xba Ⅰ digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hyg R and neo R gene transcripts were detected by RT PCR. 7 founder mice carrying hyg neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hyg R and neo R gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.

关 键 词：潮霉素抗性基因 新霉素抗性基因 转基因小鼠 小鼠胚胎成纤维细胞 

分 类 号：Q78[生物学—分子生物学]