摘要
采用PCR方法以正常中国人脐带血提取总DNA为模板，扩增出15kb的粒细胞集落刺激因子（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Kpnl位点，使其受控于2．6kb的WAP调控序列，从而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，移植至受体34母鼠，产生仔鼠85只。经PCR检测和Southern杂交分析，证实获得两只整合有人G－CSF基因的雄性鼠，整合率为2．37％。采用ELASA方法检测F1代雌鼠乳汁，结果表明成功地表达出人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。
Human genomic DNA was used as template of PCR. 1.5kb G-CSF genomic DNA was obtained using PCR amplification method. Sequence analysis showed that genomic DNA sequence of human G-CSF was correct. The vector of mammary gland expression was constructed and contained whey acid protein (WAP) 5' control region directed human G- CSF genomic DNA. In order to produce transgenic mice, 1200fertilized eggs were microninjected using WAP-G-CSF fragment. Two male transgenic mice were obtained and identified using PCR method and Southern analysis.Received November 10, 1997, revision received January 24, 1998This project supported by National '863' Programbe identified in F1 and F2 transgenic mice. Expression levels of human G-CSF in transgenic mouse milk were 120~250ng/ml.
基金
国家"863"高技术项目
