Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

Abstract

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK-cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK-cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV-and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.Techniques which allow the transfer of DNA into higher cells are the key to many studies of the function and regulation of eucaryotic genes. A number of methods have been employed to deliver DNA into cells, such as the uptake of calcium phosphate precipitates of naked DNA (13), the transfer of metaphase chromosomes (17), the injection of DNA directly into cells (18), and the fusing of DNA-carrying liposomes with cells (4). These techniques suffer from several weaknesses, including the lack of retrievability of transferred DNAs, low efficiencies of transfer, and an uncertainty concerning the physical state of the DNA which has entered the cells.An alternate approach which overcomes many of these problems has been the use of animal viruses as vectors to mediate the transfer of DNA sequences into cells (6, 7). A segment of DNA is inserted into a viral genome in the place of certain viral sequences. As a consequence, certain virus-encoded functions are lost, and the chimeric virus is rendered replication defective. Such chimeric genomes can be rescued from cells by coinfection of the cells with a competent t Present address:

Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

“…Although initial reports from a number of laboratories described low levels of globin expression in transgenic mice comparable to that in in vitro experiments (12,25,42,47), one study (43) (14). High-level transcription of viral RNA requires the viral enhancer, and therefore, as expected, near normal levels of viral RNA are seen after differentiation of MEL cells infected with pSVX3(RO) but not with pSVX,Ben-.…”

“…To date, these vectors have been used predominantly for transfer and expression of exogenous cDNA sequences under the control of viral transcriptional signals (17,20,32). In addition, several groups have reported the construction of recombinant genomes containing whole genomic gene sequences or minigenes [i.e., cDNA sequences flanked by promoter and poly(A) sites] (15, 16, 23, 24,33,40,42,48). Because it has been shown that a variety of genes contain regulatory sequences, not only 5' of the gene but also in introns (2,18,36) and intragenic sequences (8,52), we wished to determine whether, in general, intact genomic sequences could also be transferred to cells via retrovirus vectors and whether the sequences would be properly expressed within the context of an integrated provirus.…”

Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.

“…Successful gene transfer by viral infection has been demonstrated for a variety of genes inserted into retroviral vectors. These genes include selectable markers (thymidine kinase [TK] [3,27,30,34] and hypoxanthine phosphoribosyltransferase [HPRT] [20]), the dominant selectable markers for neomycin resistance (neo) (10,14,16) and xanthineguanine phosphoribosyltransferase (gpt) (10,18,23), and also nonselectable genes linked to selectable markers (21). We have tested the utility of the mutant dihydrofolate reductase (DHFR; EC 1.5.1.3) gene as a potential dominant selectable marker in retroviral vectors.…”

Cloning Vectors Derived from Animal Viruses

“…The favourite foreign gene has been the thymidine kinase (tk) gene of HSV, because expression of this gene can be selected for after infection or transfection of tk-cells. Wei et al (198l), Shimotohno & Temin (1981) and Tabin et al (1982) have used Harvey murine sarcoma virus, spleen necrosis virus and Moloney murine leukaemia virus, respectively, as vectors to construct recombinant genomes capable of transducing the HSV tk gene. The recombinant genomes thus produced are, like most strongly transforming viruses, replication-defective because the exogenous gene is inserted in place of one or more of the viral genes required for replication.…”