Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes

Abstract

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

FIG. 1.

Total cell number [mean±standard error of the mean (SEM)] in blastocysts produced by somatic cell nuclear transfer of pig, cattle, mouse, or chicken cell into enucleated pig oocytes. PA, parthenogenetically activated pig blastocyst. Letters (a, b, c, d) over the bar denote statistical difference (p<0.05).

FIG. 2.

Hatching ability (A) and total cell number per blastocyst (B) in in vitro–fertilized pig blastocysts produced from good- or poor-quality oocytes microinjected with pig or cattle mitochondria. (C) Noninjected controls. Letters (a, b, c) over the bar denote statistical difference (p<0.05).