人G—CSF基因组基因的克隆及其在转基因小鼠乳腺表达的研究

采用ＰＣＲ方法以正常中国人脐带血提取总ＤＮＡ为模板，扩增出１．５Ｋｂ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）基因的起支密码子ＡＴＧ臆的ＫｐｎＩ位点，使其受控于２．６ｋｂ的ＷＡＰ调控序列，从而构建乳腺表达载体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

Cloning of Human G-CSF Genomic Gene and Its Expression in Transgenic Mice Mammary Gland

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.