Recombinant Genomes Which Express Chloramphenicol Acetyltransferase in Mammalian Cells

TLDR

Because mammalian cells lack endogenous CAT activity and rapid assays exist, these recombinants offer a convenient system to monitor foreign DNA expression in tissue culture. The authors aimed to demonstrate the system’s usefulness by creating pSV2‑cat derivatives with partial or complete removal of the SV40 promoter. They constructed recombinant plasmids (pSV2‑cat, pSV0‑cat, and derivatives) that fuse the CAT coding sequence to SV40 promoter elements or a HindIII site, enabling expression in mammalian cells. CAT protein accumulated within 48 h in CV‑1 cells, and while deleting one 72‑bp repeat had little effect, removing an additional 50 bp from the second repeat reduced synthesis to 11 % of wild‑type levels.

Abstract

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.