Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

Abstract

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P>0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neo ( r ) genes were correctly expressed indicating that transgenic somatic cell lines and positive transgenic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cashmere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.

Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats

“…Resistant clones were obtained by G418 selection. Nuclear transfer into in vitro-matured oocytes was then performed as described previously (Guo et al, 2009). The reconstructed embryos were cultured for 48 h and the cleavage rate was calculated.…”

“…All of the transgenic cashmere goats produced were the result of random integration. Transgenic nuclear donor cells and fibroblasts isolated from cashmere goat fetuses (wild type, after a 40-day gestation) transfected with an empty vector (named CFC3) (Guo et al, 2009) were used as controls (Table 1). The transgenic cashmere goats were generated by transfection with the pDsRed2-1 vector (Catalog No.…”

Potential of Adipose-Derived Mesenchymal Stem Cells and Skeletal Muscle-Derived Satellite Cells for Somatic Cell Nuclear Transfer Mediated Transgenesis in Arbas Cashmere Goats

“…Well-formed COCs were identified by light microscopy on the basis of integrated cumulus cells, tight wrapping and having at least three layers of cumulus cells. Selected COCs were incubated in maturation medium (TCM199 [11150-059; Invitrogen Corp., Carlsbad, CA] +10% estrous goat serum [homemade] [17] +10 mM Hepes [H4034; Sigma–Aldrich, St. Louis, MO] +0.1 μg/mL 17β-estradiol [C13213100; Wako, Osaka, Japan] +10 μg/mL follicle stimulating hormone [F2293; Sigma–Aldrich, St. Louis, MO] +8 g/mL luteinizing hormone [L6420; Sigma–Aldrich, St. Louis, MO] +0.22 mg/mL sodium pyruvate [P4562; Sigma–Aldrich, St. Louis, MO]) and equilibrated at 38.5°C in an atmosphere of 5% CO 2 in air for 18 h, after which the maturation rate (number of oocytes containing the first polar body/total oocytes) was calculated. The matured COCs were treated with 0.1% hyaluronidase (H3506; Sigma–Aldrich, St. Louis, MO) to remove cumulus cells.…”

Pluripotency and Growth Factors in Early Embryonic Development of Mammals: A Comparative Approach

“…Blastocyst Human [132], mouse [133], rat [134], cattle [135], sheep [136], goat [137], rabbit [48], dog [138], buffalo [139] Early embryo Human [140], cattle [129], horse [130] Early placenta or pregnant endometrium Human [141], rat [142], rabbit [48], pig [143], dog [144], horse [127] IGF-2…”

“…Human [119], mouse [145], rat [142], cattle [146], sheep [137], pig [143], rabbit [48], goat [22], dog [138], horse [130], buffalo [139] Early embryo Cattle [129] Early placenta or pregnant endometrium Human [147], rat [142], rabbit [48], dog [144], cat [148] IGFR-1…”