利用外显子捕获技术从基因组片段中分离表达序列

IDENTIFICATION OF EXPRESSIVE SEQUENCES FROM GENOMIC FRAGMENTS BY EXONTRAPPING

本实验旨在以外显子捕获（ｅｘｏｎ－ｔｒａｐｐｉｎｇ）技术从人类５号染色体短臂（５ｐ）＂猫叫综合征＂区域分离表达序列。将来自５ｐ的基因组片段克隆进ｐＳＰＬ３剪接载体，用以转染ＣＯＳ－７细胞。经ＲＴ－ＰＣＲ扩增后，以ＵＤＧ法克隆剪接产物，并进行基因组来源、组织表达特异性及序列分析等鉴定。结果表明，基因组片段在实验系统中能被有效剪接，并得到了确为５ｐ来源的表达序列。证明外显子捕获技术能有效用于表达序列的筛选，但现有的剪接载体仍有较为严重的隐含剪接问题。

This study is aimed at identification of expressive sequences from the cri-du-cat syndrome region on the 5p by means of exon-trapping, Genomic fragments from the 5p were cloned into a splicing vector, pSPL3, and then transfected into COS-7 cells. After RT-PCR, the splicing products were cloned by UDG method, and characterized for their genomic origin, tissue-specific expression and sequence, The results indicate that the genomic fragments can be efficiently spliced in the experimental system by which expressive sequences originated from the 5p have been successfully selected,demonstrating that exon-trapping is an important technique for identification of expressive sequences, but the trapping vector,pSPL3, has the problem of cryptic splicing.