High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron.
A minigene version of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) was prepared that contained -2.3 kilobases of the 5'-flanking sequence, the first 5 exons and introns, the last 6 exons and introns, and about 2 kilobases of the 3'-flanking sequence. The gene was then used for stable transfection experiments with mouse NIH 3T3 fibroblasts. Because the products of the minigene were shorter, it was possible to compare expression of the minigene with expression of the endogenous pro alpha 1 (I) gene by Northern and Western blot analyses. The results demonstrated that the construct contained enough of the gene to obtain high levels of expression in many of the stably transfected cells. Since previous observations suggested that the first intron of the pro alpha 1 (I) gene contained important cis-regulatory elements, two versions of the minigene were prepared in which most of the first intron was deleted. Comparison of expression of the minigene with expression of two deleted versions of the same gene established that 85% of the total sequences in the first intron are not essential for high levels of expression of the gene in stably transfected mouse fibroblasts.
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
A S
AS
Olsen
A E
AE
Geddis
D J
DJ
Prockop
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002999:Clone Cells; D004247:DNA / Q000032:analysis; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D007438:Introns; D051379:Mice; D011347:Procollagen / Q000235:genetics*; D012313:RNA / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D011347:Procollagen; D012313:RNA; D004247:DNA