Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells

Abstract

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression byMo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5-to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression fromthe L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

SDF‐1/CXCL12 Enhances Survival and Chemotaxis of Murine Embryonic Stem Cells and Production of Primitive and Definitive Hematopoietic Progenitor Cells

“…E14 and CCE ESC lines are derived from 129/SV mouse embryos [25,26]. E14 and CCE ESC lines were cultured on gelatinized plates in Dulbecco's modified Eagle's medium (DMEM) with 15% ESC-qualified fetal calf serum (FCS; HyClone, Logan, UT, http://www.hyclone.com), 5.5 × 10 −2 mM β-mercaptoethanol (Gibco BRL, Carlsbad, CA, http://www.gibcobrl.com), and 10 3 U/ml leukemia inhibitory factor (LIF) (ESGRO; Chemicon International, Temecula, CA, http://www.chemicon.com).…”

Murine Embryonic Stem Cells Secrete Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis and Stimulate Colony Formation of Murine Hematopoietic Progenitor Cells

“…E14, CCE, and RI are ES cell lines derived from 0129 mouse embryos [10,11]. ES cell lines were cultured on gelatinized plates in Dulbecco's modified Eagle's medium (DMEM) with…”

Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein

“…vector constructs and packaging cell lines. As a retroviral vector, GCDNsap described here is constructed similarly to MND vector developed by Kohn's group which has the dl587rev derived primer binding site and the NCR devoid LTR whereas GCDNsap is different from MND in the virus LTR and backbone; the former is a spliced-type retroviral vector with the PCMV LTR and the latter is a LN type vector with the MPSV LTR (Robbins et al 1997). Consistent with the results obtained from the present study, MND also showed high and continued expression of the transgene in immature cells such as EC and ES cells as well as murine hematopoietic stem cells (Halene et al 1999).…”