Ganguly A, Smelt S, Mewar R, Fertala A, Sieron A L, Overhauser J, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia, PA 19107.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7365-9. doi: 10.1073/pnas.91.15.7365.
Previous studies demonstrated that type II procollagen is synthesized by HT-1080 cells that are stably transfected with constructs of the human COL2A1 gene that contain the promoter and 5' end of either the COL2A1 gene or the human COL1A1 gene. Since the host HT-1080 cells were from a human tumor line that synthesizes type IV collagen but not type II or type I procollagen, the results suggested that the constructs were integrated near active enhancers or promoters. Here, however, we demonstrate that a 33-kb construct of the COL2A1 gene containing a 5' fragment from the same gene was inserted into both alleles of the endogenous COL2A1 gene on chromosome 12, apparently by homologous recombination by a nonconservative pathway. In contrast, a similar construct of the COL2A1 gene in which the 5' end was replaced with a 1.9-kb fragment from the 5' end of the COL1A1 gene was inserted into both alleles of the locus for the COL1A1 gene on chromosome 17. Therefore, targeted insertion of the gene construct was not directed by the degree of sequence homology. Instead, it was directed by the relatively short 5' fragment from the COL1A1 gene that contained the promoter and the initially transcribed sequences of the gene. After insertion, both gene constructs were expressed from previously inactive loci.
先前的研究表明，II型前胶原由HT-1080细胞合成，这些细胞被稳定转染了包含人COL2A1基因启动子和5'端或人COL1A1基因启动子和5'端的构建体。由于宿主HT-1080细胞来自人肿瘤细胞系，该细胞系合成IV型胶原但不合成II型或I型前胶原，结果表明这些构建体整合在活性增强子或启动子附近。然而，在此我们证明，一个包含来自同一基因5'片段的33 kb COL2A1基因构建体，显然通过非保守途径的同源重组，插入到了12号染色体上内源性COL2A1基因的两个等位基因中。相比之下，一个类似的COL2A1基因构建体，其5'端被来自COL1A1基因5'端的1.9 kb片段取代，插入到了17号染色体上COL1A1基因位点的两个等位基因中。因此，基因构建体的靶向插入不是由序列同源性程度决定的。相反，它是由来自COL1A1基因的相对较短的5'片段决定的，该片段包含启动子和该基因最初转录的序列。插入后，两个基因构建体都从先前无活性的位点表达。
