异种移植模型转人a1,2岩藻糖基转移酶基因小鼠显微注射DNA片段的制备

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。

The production of microinjected DNA fragment for transgenic mouse in studying xenotransplantation

Objective To produce microinjected DNA fragment for human 1, 2-fucosyltransferase (HT) transgenic mouse. Methods The end of primer was designed to have unique EcoR I or BamH I site. A DNA fragment encoding the full length of HT gene was generated utilizing PCR, which included EcoR I (5′) and BamH I (3′) sequence end. The DNA was purified on gels and subcloned into pMD 18-T vector. HT cDNA was digested by EcoR I and BamH I, then was recovered and cloned into pCMV-MCS plasmid. The recombinant plasmid pCMV-MCS-HT cDNA was identified using PCR, digestion and sequence analysis. Pvu I and Not I digested recombinant plasmid respectively. The 2.85 kb DNA fragment was recovered and lysed in microinjected buffer. Results The recombinant plasmid pCMV-MCS-HT cDNA was constructed successfully. The 2.85 kb microinjected DNA fragment was digested and recovered. Conclusion The microinjected DNA fragment for human HT transgenic mouse can be produced by gene engineering technique, which has elements for gene expression, and can be used to produce transgenic mouse.