Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences.
We describe the construction and properties of a retroviral expression vector, designated pMV-7, designed to transfer unselected cDNAs and produce their encoded proteins in recipient cells. The vector is flanked by the long terminal repeats (LTRs) of the Moloney murine sarcoma virus (MSV) and contains the selectable drug resistance gene neo under the regulation of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Unique Eco RI and Hind III sites facilitate the introduction of sequences whose transcription is regulated by the 5' LTR. We have inserted cDNAs encoding: (i) the human lymphocyte antigen T4, (ii) the human lymphocyte antigen T8, and (iii) the murine hypoxanthine-guanine phosphoribosyl transferase (HPRT), into the pMV-7 vector. These constructions were used to transduce recipient cells to the neo+ phenotype. In each case, functional assays demonstrated that 65-92% of the neo+ clones produced the appropriate protein encoded by its corresponding cDNA. These clones were characterized further by analyzing the expression of vector-regulated transcripts. The neo+T4+ clones expressed a single full-length LTR-to-LTR transcript as detected by a T4 probe. The neo+T8+ clones, however, expressed both a full-length LTR-to-LTR transcript and an additional smaller transcript as detected by a T8 probe. This smaller transcript probably resulted from the utilization of cryptic signals which control 3' RNA processing. Furthermore, all of the neo+ clones expressed a transcript that initiated from the tk promoter, contained the neo gene, and used polyadenylation signals provided by the 3' LTR. Thus, the pMV-7 vector is capable of high-efficiency transfer and high-frequency expression of the cDNA-encoded protein.
Kirschmeier
P T
PT
Cancer Center/Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Housey
G M
GM
Johnson
M D
MD
Perkins
A S
AS
Weinstein
I B
IB
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D008980:Moloney murine sarcoma virus / Q000235:genetics; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
D011506:Proteins; D004247:DNA; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes