Hamaguchi M, Sakamoto H, Tsuruta H, Sasaki H, Muto T, Sugimura T, Terada M
Genetics Division, National Cancer Center Research Institute, Tokyo, Japan.
Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9779-83. doi: 10.1073/pnas.89.20.9779.
We have established a highly sensitive and specific exon-trapping system (SETS) with a specific plasmid vector in which an exon in a given DNA segment is identified by its ability to remain as a mature mRNA after splicing. The SETS provides us with the isolation of possible exons rapidly and easily from DNA fragments in chromosomal regions of more than 300 kilobase pairs. Genomic DNA fragments were partially digested and subsequently cloned into plasmid pMHC2, an exon-trapping vector we have constructed. These constructs were transfected into COS-7 cells, and consequent RNA transcripts were spliced in the cells. The resulting mature mRNA was harvested and amplified by using reverse transcription-PCR. Possible exons can be recognized by the sizes of PCR products and cloned into a plasmid vector. The SETS provides a direct means of cloning exons from genomic DNA of more than 300 kilobase pairs within a short period of time. Using this system, we have screened 300-kilobase-pair genomic DNA segments derived from human chromosome 11q13. Human chromosome 11q13 may contain genes responsible for human cancers, because DNA amplification is observed in several malignant tumors. We have successfully identified exon 2 of the HST1 gene and additional transcribed sequences.
我们利用一种特定的质粒载体建立了一个高度灵敏且特异的外显子捕获系统（SETS），在该系统中，给定DNA片段中的外显子可通过其在剪接后作为成熟mRNA保留的能力来识别。SETS使我们能够快速且轻松地从超过300千碱基对的染色体区域的DNA片段中分离出可能的外显子。基因组DNA片段被部分消化，随后克隆到我们构建的外显子捕获载体质粒pMHC2中。这些构建体被转染到COS - 7细胞中，随后细胞中的RNA转录本进行剪接。收获得到的成熟mRNA并通过逆转录 - PCR进行扩增。可能的外显子可通过PCR产物的大小识别出来，并克隆到质粒载体中。SETS提供了一种在短时间内从超过300千碱基对的基因组DNA中克隆外显子的直接方法。利用该系统，我们筛选了源自人类11号染色体q13区域的300千碱基对基因组DNA片段。人类11号染色体q13可能包含与人类癌症相关的基因，因为在几种恶性肿瘤中观察到了DNA扩增。我们已成功鉴定出HST1基因的外显子2及其他转录序列。
