Maloney murine leukemia virus-based, replication-defective retroviral vectors containing the neomycin resistance gene (neo) were developed to transfer the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase, into mammalian cells. To optimize gene transfer and expression, the following promoters were linked to ada: the Maloney murine leukemia virus promoter within the long-terminal repeat, the Rous sarcoma virus promoter, the thymidine kinase promoter, or the human phosphoglycerate kinase promoter. Sequences were transfected into the helper virus-free retroviral packaging \j/-2 cell line. Recombinant retroviruses were tested in CCL-1 cells, which, like most murine tissues, have low levels of alkyltransferase and are sensitive to l,3-bis(2-chloro-ethyl)nitrosourea (BCNU), and in NIH-3T3 cells, which are BCNU resistant and have high levels of alkyltransferase. Lines infected with each of the four retroviruses were selected for neo expression and found to have intact proviral integration and ada gene expression. Alkyltransferase activity was greatest with retrovirus containing the Rous sarcoma \irus-ada gene; infected NIH-3T3 cells had up to 2300 units of alkyl-transferase/mg of protein compared with 151 units/mg of protein in control cells, and infected CCL-1 cells had up to 1231 units/mg of protein compared with 33 units/mg of protein in control cells. CCL-1 cells expressing ada were more resistant to BCNU cytotoxicity than were controls. However, NIH-3T3 cells expressing ada were only slightly more resistant to BCNU than controls, possibly because most of the ada protein was cytoplasmic rather than nuclear as suggested by immunohis-tochemical stain. These studies establish a series of retroviruses containing the bacterial ada gene, which efficiently infect mammalian cells, ada expression increases nitrosourea resistance in cells with low mammalian alkyltransferase activity.
|Original language||English (US)|
|Number of pages||8|
|State||Published - Nov 1 1989|
ASJC Scopus subject areas
- Cancer Research