308 INFLUENCE OFA MATRIX ATTACHMENT REGION ON THE EXPRESSION OF BICISTRONIC VECTORS TRANSFECTED IN MAMMALIAN CELLS CULTURED IN VITRO

We investigated the effect of the 52 MAR (matrix attachment region) sequence of chicken lysozyme gene as a possible insulator for the expression of our transgenes in somatic cells to be used for nuclear transfer. With the preliminary purpose to demonstrate a possible positive effect (position or copy number) on the long-term combined expression during in vitro culture, we have created a bicistronic ubiquitous expression vector with (MAR+) or without (MAR–) MAR. The main structure of our constructs is composed of the pCAGGS promoter driving the expression of a reporter gene (enhanced green fluorescent protein, EGFP) followed by a loxable selection cassette (loxP-PGKneo). The MAR region was inserted before the selection cassette. After KpnI digestion, the resulting linearized vectors were purified and subsequently used to transfect adult porcine fibroblast cell lines using the Nucleofector system (Amaxa, Cologne, Germany). Fibroblasts were cultured in DMEM/M199 medium (1:1) + 10% FCS supplemented with 5 ng mL–1 of basic fibroblast growth factor (bFGF). In every experiment, 1 � 106 cells were transfected with 2.5 µg of linearized plasmid and selected for 3 weeks with medium supplemented with 400 µg mL–1 of Geneticin (G418 sulfate, Calbiochem, La Jolla, CA). On Day 8 of G418 selection, we analyzed 150 colonies for each treatment, using fluorescence microscopy with fluorescein isothiocyanate filters. Colonies were classified according to size (large) and cell morphology (small cells without signs of aging). In addition, colonies were classified for uniform GFP expression (uniform), patchy GFP expression (variegated), and no GFP expression (negative). Resistant colonies derived from MAR+ and MAR– vectors, respectively, had 36 (24%), 42 (28%), and 56 (37%) v. 79 (53%), 58 (39%), and 29 (19%) uniform, variegated, and negative GFP. Differences were significant for variegated and negative in MAR+ v. MAR– (chi square, P < 0.05). Thirty-six MAR+ and 42 MAR– colonies uniformly expressing GFP were transferred to 24-well plates and subjected to G418 selection until Day 22, when 7 MAR+ and 15 MAR– clones were still growing in culture. Four of seven MAR+ (57%) and 7/15 MAR– (47%) uniformly expressed high levels of GFP. In conclusion, we found that significantly fewer colonies expressed GFP with the MAR+ vector; however, within the GFP-expressing clones, expression was more uniform. Therefore, we did not find a beneficial effect of MAR sequences on expression in somatic cells during in vitro culture; however, further work is needed to investigate their effect after nuclear transfer and/or on the next generation of cloned transgenic animals.

409 EFFICIENCY OF CO-TRANSFECTION OF TRANSGENE AND Neor GENE INTO GOAT AND PIG FETAL FIBROBLASTS

Transgenic animals can be generated by nuclear transfer with genetically modified somatic cells in which the essential procedure of transgene transfection is required. Most transgene vectors are constructed to contain transgene and drug-resistant genes to enrich for somatic cells in which transgene integration has occurred. However, construction of transgene vectors along with drug-resistant genes may not be easy, due to inappropriate restriction sites. Therefore, in this study, two separate constructs, human tPA cDNA fused to β-casein promoter sequence as a transgene vector and neomycin-resistant gene (Neor) driven by PGK promoter as a drug-selectable gene, were co-transfected into pig and goat fetal fibroblast cells to estimate the efficiency of transgene transfection following G418 selection. First, goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) were tested for G418 resistance with different concentrations of G418. The pertinent concentrations of G418 were 800 µg mL−1 for GFF and 200 µg mL−1 for PFF. The linearized tPA vector and Neor gene vector were co-transfected into goat fetal fibroblasts and pig fetal fibroblasts with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). The cells were selected following exposure of 800 µg mL−1 and 200 µg mL−1 G418 for GFF and PFF, respectively, for 14 days. Cell colonies surviving G418 selection were assayed by PCR amplification with tPA-specific primers. Initially 2 × 106 GFF and PFF were transfected. Resistant colonies were counted and transferred to 24-well plates for expansion and PCR analysis. The results of co-transfection experiments are summarized in Table 1. The transfection of 2 × 106 GFF and PFF yielded an estimated 96 and 93 colonies, respectively, which survived as the G418 selection. However, 54 colonies of GFF and 39 colonies of PFF proliferated during expansion and were subjected to PCR analysis. Twenty-three and 5 of these colonies were identified to contain tPA transgene in GFF and PFF colonies, respectively. Transfection frequencies for tPA gene were 42.6% and 12.8% in GFF and PFF, respectively. These results suggest that co-transfection of transgene vector with Neor gene can be an alternative method for transfection of transgenes into fetal fibroblast cells. Table 1. Transfection efficiency of goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) following co-transfection of tPA gene and Neor gene

Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti–Epstein-Barr virus potential through both culture-dependent and selection process–dependent mechanisms

To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene–expressing donor T cells can be administered with an allogeneic T-cell–depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti–Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-γ enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer+ cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer+cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer+ cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.

Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus- producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.

Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus- producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.

Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric

Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.