TARGETED INSERTIONS OF 2 EXOGENOUS COLLAGEN GENES INTO BOTH ALLELES OF THEIR ENDOGENOUS LOCI IN CULTURED HUMAN-CELLS - THE INSERTIONS ARE DIRECTED BY RELATIVELY SHORT FRAGMENTS CONTAINING THE PROMOTERS AND THE5' ENDS OF THE GENES

Previous studies demonstrated that type II procollagen is synthesized by HT-1080 cells that are stably transfected with constructs of the hu man COL2A1 gene that contain the promoter and 5' end of either the COL 2A1 gene or the human COL1A1 gene. Since the host HT-1080 cells were f rom a human tumor line that synthesizes type IV collagen but not type II or type I procollagen, the results suggested that the constructs we re integrated near active enhancers or promoters. Here, however, we de monstrate that a 33-kb construct of the COL2A1 gene containing a 5' fr agment from the same gene was inserted into both alleles of the endoge nous COL2A1 gene on chromosome 12, apparently by homologous recombinat ion 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 fragmen t 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 i nsertion of the gene construct was not directed by the degree of seque nce homology. Instead, it was directed by the relatively short 5' frag ment from the COL1A1 gene that contained the promoter and the initiall y transcribed sequences of the gene. After insertion, both gene constr ucts were expressed from previously inactive loci.