50 FLOW CYTOMETRY-MEDIATED DETECTION OF LATE-APOPTOTIC HYPODIPLOID CELL FRACTIONS IN LIPOFECTED PORCINE ADULT DERMAL FIBROBLAST CELL LINES SELECTED FOR SOMATIC CELL NUCLEAR TRANSFER

Abstract

Analysis of nuclear DNA (nDNA) content of in vitro cultured somatic cells undergoing apoptosis became one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The cell fractions with hypodiploid (<2C) nDNA molecule number, which involve the so-called sub-G1 peak in DNA histograms are identified as late-apoptotic subpopulations. Advantage of this method is the possibility of simultaneous cell cycle measurement. The present study was conducted to investigate the preimplantation developmental outcome of porcine transgenic NT embryos reconstituted with non-apoptotic gilt ear skin-derived fibroblast cells that had been lipofected with pWAPhGH-GFPBsd gene construct. The nuclear donor cells were derived from such cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through the non-vital nDNA fluorescent dyeing and subsequent flow cytometry (FACS). Frozen/thawed fibroblast cells, which had been cultured up to a total confluency after 2–3 passages, were used for the diagnostics. The fixed dermal fibroblasts were exposed to nDNA extraction buffer for 5 min and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. Somatic cell cloned embryos, which had been created by simultaneous fusion and electrical activation, followed by delayed chemical activation of reconstructed oocytes, were cultured in NCSU-23/FBS medium for 6 to 7 days up to morula/blastocyst stages (Skrzyszowska et al. 2008 Theriogenology 70, 248–259). The FACS analysis revealed that out of all the fibroblast cells diagnosed, 94.9% were cycling and 5.1% were late-apoptotic. In turn, from among the non-apoptotic cells, an average of 92.7% were at G1/G0 stages of cell cycle, 3.1% were at S stage and 4.2% were at G2/M stages. A total of 294/348 (84.5%) enucleated oocytes were successfully fused with non-apoptotic nuclear donor cells. Out of 294 cultured NT embryos, 199 (67.7%) were cleaved. The rates of cloned embryos that reached the morula and blastocyst stages yielded 165/294 (56.1%) and 57/294 (19.4%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent lipofected adult dermal fibroblasts confirmed that the cell cycle synchronization at G1/G0 phases was highly efficient, while the frequency of late-apoptotic cells was low. It was also found that the relatively high percentages of pWAPhGH-GFPBsd transgenic blastocysts developed in vitro from NT embryos reconstructed with fibroblast cells undergoing lipofection. Furthermore, porcine cloned blastocysts exhibited approximately 100% index of reporter eGFP transgene expression, which was visually confirmed by their live-fluorescent evaluation.

This work was supported by the Scientific Net of Animal Reproduction Biotechnology.