Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus. This problem has been circumvented by segregating the viral genome into cis- and trans-acting components. By establishing helper cell lines that produce the trans-acting viral gene products, one can propagate the cis-acting component in them and harvest defective viral particles that contain only the cis-acting component. The cis-acting component can provide a useful vehicle for the highly efficient transfer of genes into target cells. The defective vector systems described to date, however, are restricted in host range to murine, avian, rat, and dog cells. We describe a helper-free vector system based entirely on an amphotropic murine virus with a wide mammalian host range, including the ability to carry out efficient gene transfer into human cells. We also describe a double mutation constructed in the trans-acting genome which reduces the frequency of replication-competent recombinant viruses to undetectable levels.

The undifferentiated embryonal carcinoma (EC) cell line PCC4 aza 1 was infected with a selectable amphotropic retrovirus. Although EC cells are generally refractory to retroviral gene expression, we found that approximately 1 of 5,000-10,000 recombinant proviruses was transcribed in these cells. Cells containing an active recombinant provirus were cloned and expanded. Nucleic acid analysis revealed approximately one intact provirus per cell. Viral RNA levels were different for each cell clone examined [between 0.05% and 0.5% of the poly(A)+ RNA], and transcription was initiated and terminated in the long terminal repeats as in permissive differentiated cells. The cells retained an undifferentiated phenotype and remained positive for stage-specific embryonic antigen 1 and negative for H-2 surface antigens. The cells retained the block to the expression of other proviruses integrated at other chromosomal locations. The data suggest that a cisacting genetic element, at or near the chromosomal site of integration, or mutations in the viral control elements themselves may be responsible for provirus expression in these cells.The ability to transfer cloned genes into preimplantation embryos has opened the way for the study of gene expression in the maturing organism. Unfortunately, much of the early work has suggested that many cloned genes do not carry the information necessary to guarantee their proper expression during differentiation (1-4). Similarly, the expression of cloned retroviral genes introduced into preimplantation embryos seems to depend on the chromosomal site of integration (5). Most retroviral genomes introduced in this way are not expressed at all and become highly methylated (5).Undifferentiated embryonal carcinoma (EC) cells provide a useful model system for studying early gene expression. As in early embryos, retroviral genomes are not expressed in undifferentiated EC cells (6-8). Yet EC cells present no block to viral penetration, reverse transcription, or viral DNA integration (9, 10). Although newly integrated proviruses become methylated in EC cells (10), this modification occurs several days after integration, suggesting that methylation may be a result, not a cause, of provirus inactivity (11,12). Thus, a provirus that has integrated into a transcriptionally active region of a chromosome might escape inactivation-at least while that region remained transcriptionally active.With this in mind, we infected undifferentiated EC cells with a selectable recombinant retrovirus (13) Fig. 1).This recombinant genome replicates in the presence of amphotropic helper virus. Reverse transcriptase levels were measured as described (16).Infections. Culture supernatant containing defective cistorneo particles was adjusted to 8 ,ug of Polybrene per ml and passed through a 0.45-gm filter. The filtrate was added to plates of NIH 3T3 or PCC4 target cells for 2 hr. The plates were then supplemented with regular culture media. Certain PCC4 lines containing cistomeo proviruses were superinfected with amphotr...

Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus. This problem has been circumvented by segregating the viral genome into cis- and trans-acting components. By establishing helper cell lines that produce the trans-acting viral gene products, one can propagate the cis-acting component in them and harvest defective viral particles that contain only the cis-acting component. The cis-acting component can provide a useful vehicle for the highly efficient transfer of genes into target cells. The defective vector systems described to date, however, are restricted in host range to murine, avian, rat, and dog cells. We describe a helper-free vector system based entirely on an amphotropic murine virus with a wide mammalian host range, including the ability to carry out efficient gene transfer into human cells. We also describe a double mutation constructed in the trans-acting genome which reduces the frequency of replication-competent recombinant viruses to undetectable levels.