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

Abstract

The expression of Moloney murine leukemia virus and vectors derived from it is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have developed a retroviral vector, the murine embryonic stem cell virus (MESV), that is active in embryonal carcinoma and ES cells. MESV was derived from a retroviral mutant (18,19). In addition, MPSV and PCMV contain point mutations within the leader sequences that are also major determinants in the expanded permissiveness of these vectors, at least in EC cells (13,15). In this study we have analyzed the expression of PCMV vectors in ES cells. We found that the enhancer region of PCMV was functional in ES cells. However, sequences located within the 5' untranslated region of the viral genome completely abolished viral expression in ES cells. Replacement of this region by functionally equivalent sequences obtained from the dl-587rev virus (20) allowed LTR-mediated expression of retroviral genomes in ES cells, independent of the chromosomal site of integration. MATERIALS AND METHODSCell Culture and Viral Infections. 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). 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.For DNA and RNA analysis of neomycin-resistant (neoR) NIH 3T3 and ES cells, cells were infected with viruscontaining cell supernatant and selected in G418-containing medium (0.2 mg/ml). The neoR clones were pooled after 10-12 days in selective medium and expanded. For DNA and RNA analysis of infected and nonselected cells, NIH 3T3 and CCE cells were infected six times with virus supernatants during a 1-week period. Cells were then kept in culture for 3 days before analysis.

Double-copy bicistronic retroviral vector platform for gene therapy and tissue engineering: application to melanoma vaccine development

“…15 25,26 Consequently, safety-modified versions of the ure 1. In MSCVnlslacZ, the nlslacZ gene is expressed from the retroviral LTR and the selectable neo marker gene is MSCV backbone were created for gene therapeutics involving human hematopoietic cells.…”

“…5 On the other alleviate the transcriptional block experienced by MoMLV-based retroviral vectors in undifferentiated hand, production of individualized autologous genetransduced melanoma vaccines ex vivo is a labor-intencells. 15,16 Transcriptional extinction has also been seen with MoMLV-based LTRs in vivo. 17,18 The melanoma cell sive, time-consuming and costly endeavor.…”

“…34 In preclinical studies with the B-78 murine melanoma observed in some instances. [15][16][17][18] Even if an ex vivo selection step can be employed, the selection strategy does not model, we found that the combination of interleukin-6 (IL-6) and soluble IL-6 receptor was more effective in ensure maintenance of expression of the therapeutic gene in vectors having this double-promoter configuration.…”

High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo

“…9 Expression of mCD8 was compared in HuH-7 cells between the retroviral vectors, MO1/Lyt and MO3/Lyt, which were identical, except the leader sequences. 18 Leader sequences of MO1/Lyt contained the PBS of mouse embryonic stem cell virus (MESV) (PBS for tRNA Gln ), while that of MO3/Lyt contained the PBS of MoMLV (PBS for tRNA Pro ) (Figure 3a).…”

“…A series of chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed, which contained the U3 regions of spleen focus-forming virus (SFFVp), 7 Myeloproliferative sarcoma virus (MPSV), 8 PCC4-cell-passaged MPSV (PCMV) 9 or Moloney murine leukemia virus (MoMLV) in the same plasmid backbone. These plasmid DNAs were transfected into the cell lines derived from differentiated hepatocellular carcinomas (HuH-7 10 and PLC/PRF/5 11 ), from an undifferentiated hepatocellular carcinoma (HLE 12 ), from a hepatoblastoma (HepG2 13 ), from a cholangiocelluler carcinoma (KMC-1 14 ) or from a combined hepatocholangiocarcinoma (KMCH-1 15 ).…”