小鼠LIF编码基因的克隆与真核表达载体pcDNA3．1-LIF的构建

Cloning of mouse full-lenth LIF gene and construction of eukaryotic expression vector pcDNA3.1-LIF

目的:通过克隆小鼠LIF编码基因,构建pcDNA3.1-LIF真核表达载体,为下一步建立转基因动物模型奠定基础。方法:采用ICR雌性小鼠子宫组织,提取总RNA,运用RT-PCR技术,扩增出小鼠LIF基因全长编码序列,采用同源重组方法构建具有新霉素抗性的小鼠LIF真核表达载体pcDNA3.1-LIF。结果:通过PCR获得了约612bp大小的特异性cDNA片段。经核苷酸序列分析,扩增得到的片段与小鼠LIFcDNA序列同源性为100%。经PCR及酶切鉴定筛选阳性克隆菌,表明LIF编码序列正确连入pcDNA3.1载体中。结论:成功克隆了小鼠LIF编码基因,并构建了真核表达载体pcDNA3.1-LIF。

Objective To construct an eukaryotic expression vector pcDNA3.1-LIF by cloning mouse LIF genes and provide basis for establishment of transgenic animal models. Methods Total RNA was extracted from the tissue of the ICR mouse uterus . The full-lenth LIF gene of the mouse was amplified by RT-PCR and. an eukaryotic expression vector peDNA3.1-LIF with neomycin resistant was constructed by homologous recombination. Results The amplfied fragments was LIF eDNA about 612 bp, it had 100% homogeneity with mouse LIF cDNA sequence by nueleotide sequence analysis. An eukaryotie expression vector pcDNA3. 1-LIF with neomycin resistant of the mouse was successfully constructed by the identification of enzyme digestion. Conclusion The mouse LIF gene is successfully cloned and an eukaryotic expression vector pcDNA3. 1-LIF is successfully constructed.