转基因小鼠乳腺表达G-CSF的研究

Expression of human G-CSF in mammary gland of transgenic mice

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。

Chinese genomic DNA was used as template of PCR, 1. 5 kb G-CSF genomic DNA was obtained by using PCR amplification method. Sequence analysis showed that genomic DNA sequence of coding G-CSF protein was correctly. The vector of mammary gland expression was constructed that contained whey acid protein (WAP) 5' control region directed human G-CSF genomic DNA. In order to produce transgenic mice, 1200 fertilized eggs were micronin-jected using WAP-G-CSF fragment. Two male transgenic mice were obtained by microninjection fertilized eggs of mice and identified by using PCR method and Southern analysis. 2. 37% of G CSF gene was integrated in mice. Foreign gene could also be identified in F1 and F2 transgenic mice. Expression levels of human G-CSF in transgenic mice milk were 120-250 ng/ml.