利用酵母人工染色体(YAC)减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

RECONSTRUCTION OF LARGE GENOMIC SEGMENT CODING FOR HUMAN IMMUNOGLOBULIN κ CHAIN BY MEIOTIC HOMOLOGOUS RECOMBINATION OF YEAST ARTIFICIAL CHROMOSOME

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。

Meiotic homologous recombination between overlapping yeast artificial chromosome (YAC) can be used to reconstruct larger genomic segment in vivo. We have screened CEPH and Olson human genomic YAC libraries and obtained two YAC clones containing the large genomic segments coding for partial human Igκ. In order to reconstruct whole gene cluster of human Igκ,two yeast cells containing the different YAC described over were mated and induced to undergo meiotic division and sporulation . Analyzing the results of PCR and Southern blotting, we obtained a clone containing a single recombinant YAC with 400kb in length, which spans almost the entire gene cluster of human Igκ, including 32 Vκ,5Jκ, constant domain genes. Besides, we have improved and simplified the traditional method of meiotic recombination between overlapping YACs, and the results indicate the feasibility of this method to reconstruct any larger intact genomic segments.