Retroviral mutants efficiently expressed in embryonal carcinoma cells.

Abstract

The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418-resistance with NeORMPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower.To isolate NeoRMPSV mutants that are efficiently expressed in PCC4 cells, G418-resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia'virus. Viral isolates (PCMV-5 and -6; PCMV = PCC4 cell-passaged Neo".MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR-MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells.

Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.

“…The ES cell line CCE was obtained from M. Evans (Department of Genetics, Cambridge University, Cambridge, U.K.) at passage 10, grown on mitomycin-treated primary fibroblast feeder layers, and frozen at passage 11. The undifferentiated state of the cells was monitored by immunofluorescence as described (17). For viral infections ES cells were grown on gelatin-coated plates in buffalo-rat-liver-cell-conditioned medium (21).…”

“…For viral infections ES cells were grown on gelatin-coated plates in buffalo-rat-liver-cell-conditioned medium (21). Infection of fibroblasts and ES cells was performed as described (17). Neomycin-resistant colonies were counted after 12-14 (NIH 3T3 and CCE) or 8-9 days (ES.D3) in selective medium.…”

“…Hybridization of the riboprobes to total cellular RNA, treatment of the hybrids, and analysis of the radiolabeled RNA were performed according to standard protocols (24). The efficiency of neoR transfer of mos--neoR-PCMV was compared with that of mos--neoRMPSV, which does not confer G418 resistance to PCC4 cells (17,18) and, therefore, was not expected to express the neoR gene in ES cells. The efficiency of neoR transfer to fibroblasts was similar for both viruses (Table 1).…”

“…The first of such mutants, selected for transformation of hematopoietic precursor cells, was the myeloproliferative sarcoma virus (MPSV) (16). MPSV vectors are expressed in the EC cell line F9 but not in other embryonic cell lines (7,17). Direct selection for expression in PCC4 cells generated a mutant of MPSV, the PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV), which confers G418 resistance to NIH 3T3, F9, and PCC4 cells with high efficiencies (17,18).…”

“…MPSV vectors are expressed in the EC cell line F9 but not in other embryonic cell lines (7,17). Direct selection for expression in PCC4 cells generated a mutant of MPSV, the PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV), which confers G418 resistance to NIH 3T3, F9, and PCC4 cells with high efficiencies (17,18). Molecular analysis of the genomes of MPSV and PCMV revealed multiple base-pair (bp) substitutions within the viral long terminal repeats (LTRs), which have been shown to be essential for the host-range expansion of these viruses (18,19).…”