64 PRODUCTION OF TRANSGENIC CLONED PIGS BY MEANS OF SOMATIC CELL NUCLEAR TRANSFER USING KUSABIRA-ORANGE GENE-TRANSFECTED CELLS

Cloned pigs that express cell markers such as fluorescent proteins (Vintersten et al. 2004 Genesis 40, 241–246) are useful in biomedical research in areas such as cell/tissue transplantation and regenerative medicine. In this study, we attempted to produce transgenic cloned pigs from porcine fetal fibroblasts which carry the gene of red fluorescent protein, humanized Kusabira-Orange (huKO). We examined whether huKO-transfected cells are suitable as nuclear donors for somatic cell cloning, and whether red fluorescence can be detected in the cloned embryos. We used porcine fetal fibroblasts transfected with the huKO gene and a retroviral vector as the nuclear donor cells. Non-transfected cells were used as the control. Cumulus–oocyte complexes collected from slaughterhouse ovaries were in vitro-matured in NCSU23 medium to produce recipient oocytes. Nuclear transfer was conducted using a previously reported method (Kurome et al. 2003 Cloning Stem Cells 5, 367–377); the following parameters which determine the overall efficiency of nuclear transfer were investigated: (1) fusion rate between the donor cells and recipient oocytes, (2) rates of normal cleavage and blastocyst formation of the NT embryos, and (3) cell numbers in each blastocyst. A DC pulse (190 V mm-1) was used for electric fusion, and NCSU23 or PZM-5 medium was used for culturing the cloned embryos. The NT embryos on Day 7 were examined under a fluorescence microscope (G excitation) in order to evaluate the expression of red fluorescence. Some cloned embryos at the 1- to 8-cell stage (Day 1 or 2) were transferred into oviducts of estrus-synchronized recipient gilts. There was no significant difference (chi-square test) between the huKO and the control groups in the rate of fusion (132/151, 87.4% vs. 134/147, 91.2%, respectively) and cleavage rate (78/132, 59.1% vs. 86/134, 64.2%, respectively). A significantly greater percentage of huKO cell-derived embryos developed into blastocysts than did control cell-derived embryos (37/132, 28.0% vs. 20/134, 14.9%, respectively; P < 0.05). However, there was no significant difference in the blastocyst cell numbers (Student's t-test: 48.6 ± 4.8 vs. 42.3 ± 4.9, respectively). Of the 132 NT embryos, 116 (87.9&percnt;) expressed red fluorescence. The percentage of blastocysts expressing red fluorescence was 94.6&percnt; (35/37). These results demonstrate that it is possible to obtain cloned blastocysts at a high rate by nuclear transfer of cells that have been transfected with huKO using a retroviral vector, and that it is possible to observe the expression of red fluorescence in cloned embryos. With respect to the cloned embryos that did not show expression of red fluorescence, we hypothesize that this was the result of a small proportion (