Viable Transgenic Goats Derived from Skin Cells

Abstract

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

“…Based on the morphological features of the collected COCs, one of the alternatives attempted to improve results was the use of in vivo-matured oocytes. However, this issue seems to be controversial, since while Reggio et al (2001) found no differences between in vitro-and in vivo-matured oocytes, showing that both oocyte sources were similar in competence to support in vivo development after goat cloning, whereas Behboodi et al (2004) and Martins et al (2016) did not obtain pregnancies after the transfer of cloned embryos using in vitro-matured oocytes. Unlike Reggio et al (2011), our experiment found that nuclear maturation rate was higher in the group of in vitro-matured oocytes than the in vivo-matured counterparts.…”

Recent Advances in Reproductive Biotechnologies in Sheep and Goat

“…In most cases, the oocytes are collected from abattoir ovaries using the slicing method and undergo IVM for 17h (goat: [59][60][61], Sheep: [34,27]). Some laboratories that conduct goat SCNT programs preferred in vivo derived oocytes either following ovulation [62,63] or LOPU [64,65]. This may be due to the decreased availability of goat abattoirs in different countries or the higher success rates from in vivo matured oocytes.…”

Production of Boer goat (Capra hircus) by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes

“…Since 1997 when the first SCNT sheep was announced (Wilmut et al, 1997), offspring have been produced in a number of different species (Vajta and Gjerris, 2006). The cloning of goats by SCNT has also been reported (Baguisi et al, 1999;Keefer et al, 2001Keefer et al, , 2002Reggio et al, 2001;Zou et al, 2001Zou et al, , 2002Behboodi et al, 2004;Chen et al, 2006;Lan et al, 2006;Shen et al, 2006).…”

“…Sci., 54, 2009 (10): 448-460 Original Paper Previously, goat cloning by somatic cell nuclear transfer was reported (Baguisi et al, 1999;Zou et al, 2001Zou et al, , 2002Behboodi et al, 2004;Chen et al, 2006;Lan et al, 2006;Shen et al, 2006). But the success rate has never been desirable.…”