Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells.

The aim of this study was to determine the developmental ability of cloned embryos derived from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, differentiated cells from NS and AFS cells, fetal fibroblast (FF) cells, adult fibroblast (AF) cells and mammary gland epithelial (MGE) cells. NS, AFS and FF cells were isolated from embryonic day 30 porcine fetus, AF and MGE cells were isolated from adult pig. NS and AFS cells were induced to differentiate into different cell types, respectively. Stem cells and their differentiated cells were harvested for analysis of the markers using reverse transcription PCR. NS and AFS cells, their differentiated cells, FF, AF and MGE cells were used for nuclear transfer, respectively. A total of 100 two-cell stage cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that the neurospheres were positive for the undifferentiated neural cell marker, Nestin and NS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, Desmin, CD-90, Nanog and Sox2. AFS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Nanog, Sox2, Oct4, Desmin and CD-90. Both NS and AFS cells were differentiated into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, NSE+ and MAP2+), adipocyte (LPL+ and PPARγ-D+), osteoblast (Osteonectin+ and Osteocalcin+), myocyte (myf-6+ and myoD+) and endothelium (CD31+, CD34+, CD144+ and eNOS+). Four cloned fetuses (28 and 32 days) derived from NS and AFS cells were obtained. The developmental potential of the cloned embryos derived from stem cells (NS and AFS cells) were higher (P < 0.05) than that of the cloned embryos derived from somatic cells (the differentiated cells from NS and AFS cells, FF cells, AF cells and MGE cells), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.

Neurogenic differentiation of EGFP gene transfected amniotic fluid-derived stem cells from pigs at intermediate and late gestational ages.

The aims of this study were (i) to determine whether amniotic fluid-derived stem cells (amniotic fluid-derived stem; AFS cells) could be isolated from pigs at intermediate and late gestational ages, and (ii) to determine if these AFS cells could be differentiated in vitro into neural lineages following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Amniotic fluid-derived stem cells were isolated from embryonic day 60 and day 110 porcine amniotic fluid respectively, and transfected with EGFP gene using lipofection. The transfected AFS cells were induced to differentiate into cells of neuronal lineages. Markers associated with undifferentiated AFS cells and their neural derivatives were tested by polymerase chain reaction. The results demonstrated that porcine AFS cells could be isolated at intermediate and late gestational ages and that transfected AFS expressed EGFP and could be induced to differentiate in vitro. Undifferentiated AFS cells expressed POU5F1, THY1 and SOX2, while following differentiation cells expressed markers for astrocytes (GFAP), oligodendrocytes (GALC) and neurons (NF, ENOS and MAP2).