Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes.
A procedure is described for stably expressing cloned genes at high levels in vertebrate cells and for obtaining these genes in high-titer virus preparations. The process uses retroviral vectors and mixtures of two "packaging cell lines" that incorporate retroviral genomes into virions with different host-range envelopes. In these cocultures, interference barriers to superinfection are overcome, retroviral vectors can replicate in the absence of a transmissible helper virus, and the cells become infected with multiple copies of the provirus that contains the cloned gene. This procedure was used to amplify expression of the membrane glycoprotein that is encoded by Friend spleen focus-forming virus, a retrovirus that is replication defective in other cell cultures. Amplifications were measured at the DNA provirus, RNA, and protein levels. In addition, the human growth hormone gene was inserted into retroviral vectors and we observed amplifications of growth hormone synthesis and secretion. The amplified growth hormone was properly processed as indicated by immunoblot analyses. A vector is described (pSFF) that is exceptionally active in coculture amplification.
Bestwick
R K
RK
Department of Biochemistry, School of Medicine, Oregon Health Sciences University, Portland 97201.
Kozak
S L
SL
Kabat
D
D
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics; D007118:Immunoassay; D009052:Leukemia Virus, Murine / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013155:Spleen Focus-Forming Viruses / Q000235:genetics* / Q000254:growth & development; D014162:Transfection
D004279:DNA, Viral; D013006:Growth Hormone