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.
Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5min (5μM) and with 6-dMAP (3mM) for 3h followed by culturing in SOF+0.8% BSA for 7 to 8 days at 38°C with 6% O2, 5% CO2, and 89% N2 in a modular incubator chamber. The experimental group was treated with cytochalasin B (5μg/mL)in the culture media for 30 to 45min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol+20% ethylene glycol in OCM, respectively) for 5min each, followed by vitrification solution 3 (25% glycerol+25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25cc cryopreservation straw, followed by an air bubble, and then a 0.25M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5s at room temperature, then in 22°C water for 15s. After thawing, the contents of the straw were expelled, mixed, held for 5min, and finally placed in OCM for 5min. Recovered embryos were placed in SOF+20% FBS and incubated at 38°C with 5% CO2 in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts.
Table 1
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.