Cloning of Asian yellow goat (C. hircus) by somatic cell nuclear transfer: Telophase enucleation combined with whole cell intracytoplasmic injection

Abstract

Our and other previous studies have shown that telophase enucleation is an efficient method for preparing recipient cytoplasts in nuclear transfer. Conventional methods of somatic cell nuclear transfer either by electro-fusion or direct nucleus injection have very low efficiency in animal somatic cell cloning. To simplify the manipulation procedure and increase the efficiency of somatic cell nuclear transfer, this study was designed to study in vitro and in vivo development of Asian yellow goat cloned embryos reconstructed by direct whole cell intracytoplasmic injection (WCICI) into in vitro matured oocytes enucleated at telophase II stage. Our results demonstrated that the rates of cleavage and blastocyst development of embryos reconstructed by WCICI were slightly higher than in conventional subzonal injection (SUZI) group, but no statistic difference (P > 0.05) existed between these two methods. However, the percentage of successful embryonic reconstruction in WCICI group was significantly higher than that in SUZI group (P < 0.05). After embryo transfer at 4-cell stage, the foster in both groups gave birth to offspring. Therefore, the present study suggests that the telophase ooplasm could properly reprogram the genome of somatic cells, produce Asian yellow goat cloned embryos and viable kids, and whole cell intracytoplasmic injection is an efficient protocol for goat somatic cell nuclear transfer.

Production of Sei Whale (Balaenoptera borealis) Cloned Embryos by Inter- and Intra-Species Somatic Cell Nuclear Transfer

“…However, for reconstruction of sei whale iSCNT embryos, ICI seems to be superior to SUZI as evidenced by the higher fusion rates in embryos reconstructed by ICI. Similarly, Chen et al [27] found both embryo reconstruction methods to be effective with respect to embryo developmental potential in Asian yellow goats (C. hircus), although ICI had higher fusion rates than those of SUZI. This indicates that cell fusion conditions including the cell fusion medium should be optimized to obtain satisfactory fusion rates in sei whale iSCNT embryos reconstructed by SUZI.…”

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

“…Although cloning by SCNT is well established in goats, with birth rates similar to those found in other species, there are no reports of cloned goats born in the tropics of the world, between parallels 30 o N and 30 o S, with all cloned goats born in countries under temperate or subtropical climates (Baguisi et al, 1999;Keefer et al, 2001;Reggio et al, 2001;Ohkoshi et al, 2003;Lan et al, 2006;Chen et al, 2007;Folch et al, 2009;Akshey et al, 2010;Colato et al, 2011;Liu et al, 2011;Nasr-Esfahani et al, 2011;Wells et al, 2011;Meng et al, 2012;Zhou et al, 2013;Yuan et al, 2014;Feng et al, 2015;Hosseini et al, 2015;Zhang et al, 2015;Yang et al, 2016a;Bai et al, 2017). This fact may be associated with the low productive and reproductive indexes of goat herds in tropical countries, such as in the Brazilian Semi-Arid region, where the annual birth rate in goats does not exceed 20% (Guimarães, 2006).…”

“…Normally, better quality oocytes, usually grades I (GI) and II (GII), are selected for in vitro maturation (Chen et al, 2007;Tang et al, 2011). When analyzing data regarding the quality of immature COCs in this study, less than 20% of selected COCs were rated as GI and GII, with a large portion of the COCs lacking or having little cumulus cells vestment.…”

“…For the reconstruction of ZI embryos, the nucleus donor cells (MSC, AF or FF) were either transferred by micromanipulation to the perivitelline space of enucleated goat oocytes (reconstruction by cell fusion, CF), or were injected directly into the ooplasm (reconstruction by cell injection, CI), according to Keefer et al (2001) and Chen et al (2007), respectively. Prior to injection into the ooplasm, cells were consecutively pipetted with a 12 µm reconstruction pipette until a deformation of the cell membranes was visible.…”

Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs

“…Genomic DNA was extracted from tissue collected from the three NT-derived kids that died during the perinatal period and the recipient goats, and analyzed using five microsatellite markers (Table 1) using methods described elsewhere (Chen et al, 2007). The microsatellite alleles for the NT-derived offspring were compared with those from transfected goat donor cells and contrasted with those from the recipient goats that carried the respective pregnancies.…”