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目的制备转人琢1,2岩藻糖基转移酶(HT)基因小鼠显微注射DNA片段。方法引物两端设计酶切识别位点,聚合酶链反应(PCR)扩增HTcDNA全长序列,两端含有EcoRⅠ和BamHⅠ酶切识序列;回收HTcDNA片段并与PMD18-T载体连接,EcoRⅠ和BamHⅠ双酶切纯化质粒鉴定;EcoRⅠ和BamHⅠ双酶切pMD18-HTcDNA重组质粒和pCMV-MCS质粒,回收HTcDNA片段和pCMV-MCS质粒片段,进而连接,转化感受态细菌,纯化质粒对其进行酶切、PCR和测序鉴定;PvuⅠ和NotⅠ依次单酶切重组质粒pCMV-MCS-HTcDNA,回收大小约2.85kb片段,溶于适量显微注射用缓冲液。结果成功构建了重组质粒pCMV-MCS-HTcDNA,酶切回收了2.85kb的显微注射DNA片段。结论通过基因工程技术可以获得转人HT基因小鼠显微注射DNA片段,包含基因表达元件,可以用于显微注射法建立转人HT基因小鼠。
Objective To prepare DNA fragments transfected by micro-injection of fucosyltransferase (HT) gene mouse. Methods The full-length HT cDNAs were amplified by polymerase chain reaction (PCR), and the EcoRⅠ and BamHⅠ sequences were digested with restriction endonucleases. The HTcDNA fragments were recovered and ligated with PMD18-T vector. EcoRⅠ and BamHⅠ Double digestion and purification of plasmid identification; EcoR Ⅰ and BamH Ⅰ double digestion pMD18-HTcDNA recombinant plasmid and pCMV-MCS plasmid recovered HTcDNA fragment and pCMV-MCS plasmid fragment, and then connect and transform competent bacteria, the plasmid was digested, PCR and sequencing. The recombinant plasmid pCMV-MCS-HTcDNA was digested with Pvu I and Not I in sequence, and a fragment of about 2.85 kb was recovered and dissolved in appropriate microinjection buffer. Results The recombinant plasmid pCMV-MCS-HTcDNA was successfully constructed and the 2.85kb microinjected DNA fragment was recovered by enzyme digestion. Conclusion Microinjection of DNA fragments, including gene expression elements, into transgenic HT mice can be obtained by genetic engineering and can be used for the microinjection to establish transgenic HT mice.