人地中海贫血β654基因作为外源基因用于转基因的制备方法

A Technique for Constructing β654-globin Gene as Exogenous Gene to Produce Transgenic Animal Models

目的探讨人地中海贫血β654基因为外源基因用于转基因小鼠制作时,外源基因的制备方法,为进一步制作转基因小鼠打下基础。方法采用反向点杂交法确诊人地中海贫血β654纯合子DNA,以PCR法扩增获得人地中海贫血β654基因,分离纯化后,通过连接酶反应将其克隆入含βLCR调控序列的基础载体pBGT51质粒中,经PCR扩增、酶切、反向点杂交及DNA测序鉴定重组质粒,用EcoRⅤ酶切获得转基因所用的外源基因。结果将人地中海贫血β654基因作为外源基因克隆入基础载体pBGT51质粒中构建了重组质粒βBGT51,用EcoRV酶切获得含人β654基因及βLCR调控序列的6.5kb的DNA片段,制备了可用于显微注射转基因的外源基因。结论采用该方法构建的含人地中海贫血β654基因的重组质粒,可获得用于显微注射转基因的外源基因。

Objective To explore the techniques for constructing β654 globin gene as an exogenous gene to produce transgenic mice. Methods To amplify β654 globin gene 3374 bp and clone into basic plasmid pBGT51, a 9 3 kb recombinant plasmid βBGT51 was constructed containing human β globin gene, βlocal controlled region and basic eletment. Recombinant plasmid βBGT51 were identified by polymerase chain reaction, enzyme restriction, reverse dot blot hybridization and DNA sequencing. Results Recombinant plasmid βBGT51 was constructed containing human β globin gene, β local controlled region, a 6 5 kb exogenous gene was obtained by EcoR V enzyme restriction for production of transgenic animals. Conlusions Recombinant plasmid βBGT51 may be obtained by this technique and serve as a 6 5 kb exogenous gene for microinjection to produce transgenic animals.