successfully used hygromycin and histidinol (43) selections with the 293G and 293GPG cells.
Most of the problems related to the generation of RCV by different retroviral packaging cell lines can be practically eliminated through the use of specific vectors (34, 35) and/or the practice of isolating clonal virus producing cell lines and screening the cell lines for helper. Nevertheless, several of the design features of the 293GPG packaging cell line may also provide at least some theoretical advantages over existing cell lines with regard to the possibility of release of RCV. One problem with all existing murine-based packaging cell lines is the presence of both endogenous retroviral DNA sequences (31, 33) and retroviral-like RNAs (44) that are efficiently packaged and transmitted to cells, and may contribute to the generation of helper virus under certain conditions. Another issue relates to the potential of different packaging cell lines to give rise to helper virus due to the overlap of sequences between the particular vector used and the precise packaging sequences present in the packaging cell line (34, 35). A more global inherent defect in the design of all murine and human-derived packaging cell lines is the ability of retroviral RNAs which lack packaging sequences to be packaged, albeit at low efficiency, and transmitted to cells (40). Even in the case of third generation packaging cells (15), it has been possible to observe the transmission of viral packaging functions to recipient cells. Cosset et al., for example, have recently documented and quantitated the transfer of both gag and env encoding genomes derived from both a packaging construct used to generate ΨCRIP cells (15) and a construct used in the generation of the human-derived FLY cell line (40), another third generation packaging cell line. In our laboratory’s unpublished studies with ΨCRE/ΨCRIP cells and the parental MFG vector, which contains an extended gag ORF (19), we have also obtained data consistent with the transfer of packaging functions and the possible emergence of helper virus in the context of high titer cross-infections employed to generate complex populations of virus-producing cells.
The design of the 293GPG cells may be relevant to each of the above issues. In the construction of the 293GPG cell line, we have used only the precise viral sequences necessary to encode gag-pol and an expression vector that utilizes totally nonretroviral sequences. We have also utilized totally nonretroviral sequences to provide for the host range of the virus produced from the cells rather than use conventional retroviral env gene expression constructs. Depending on the vector used in conjunction with 293GPG cells, this design feature of the cells may reduce the probability of undesirable recombination events. More significantly, it is hopeful that the removal of all extraneous viral sequences in the transcript used to express gag-pol and the use of nonretroviral transcripts encoding VSV-G will reduce the efficiency with which those transcripts can be packaged and transmitted to cells, relative to that which occurs with conventional third generation packaging cell transcripts. This hypothesis will need to be tested directly.
We thank Dr. Jean Schaffer for helpful discussions and critical review of this manuscript. D.S.O. is supported by a National Institutes of Health Physician Scientist Award (HL02910). Support for this work was also provided for by a Program of Excellence in Molecular Biology Grant from the National Heart, Lung, and Blood Institute (HL41484).
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