Targeted insertions of two 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 the 5' 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 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.
Ganguly
A
A
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia, PA 19107.
Smelt
S
S
Mewar
R
R
Fertala
A
A
Sieron
A L
AL
Overhauser
J
J
Prockop
D J
DJ
D016475:3T3 Cells; D000483:Alleles; D000818:Animals; D002881:Chromosomes, Human, Pair 12; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics*; D003360:Cosmids; D004247:DNA; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D003094:Collagen; D004247:DNA