Development and Gene Expression of Porcine Cloned Embryos Derived from Bone Marrow Stem Cells with Overexpressing Oct4 and Sox2

Abstract

The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors.

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

“…The rates of SCNT blastocyst formation in bovine and mouse were more than 25% and were comparatively determined as higher efficiency than those of porcine SCNT (10%–17%) during in vitro pre‐implantation period. Furthermore, total cell number of porcine SCNT blastocysts consisted of 26–50 cells was generally lower than values from bovine or mouse SCNT blastocysts, which was more than 100 cells or approximately 60 cells, respectively (Chen et al., ; Costa‐Borges et al., ; Kumar et al., , ; Lee et al., ; Pan et al., ; Seaby et al., ). But in the present study, even after supplementing with autologous ooplasm into SCNT (AOT) embryos, no significant increases in the values of cleavage rate, blastocyst formation, hatched blastocyst and total cell number in comparison with SCNT embryos were observed.…”

“…During assessment of total cell number in AOT group, the distribution of transferred ooplasm labelled with MitoTracker Green FM was observed under the fluorescence microscope (Leica, Heerbrugg, Switzerland) as well. And apoptosis‐related DNA fragmentation in blastocyst was analysed by TUNEL using in situ Cell Death Detection Kit (Roche, Basel, Switzerland) following manufacture's instruction and previous article (Lee et al., ). To evaluate the number of apoptotic body via TUNEL staining, data were obtained from five replicates (each n = 10).…”

Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

“…For the production of in vivo and in vitro embryos, IVO and embryo manipulations for PA, IVF and SCNT were performed as previously described (Kumar et al 2007(Kumar et al , 2012(Kumar et al , 2013Lee et al 2014). For preparation of IVO embryos, oestrous sows were fertilised by artificial insemination twice with a 12-h interval between inseminations.…”

“…All embryos produced in vitro, including IVF, PA and SCNT embryos, were then cultured in sets of 30 embryos per 30-mL droplet of PZM5 medium supplemented with 3 mg mL À1 BSA, 20 mg mL À1 Eagle's amino acids in basal medium (BME), 10 mg mL À1 non-essential amino acids (NEAA) for 4 days at 38.58C in a humidified atmosphere of 5% CO 2 in air and then further cultured in the same medium supplemented with 10% FBS for an additional 2 days, in the same way as previously described (Kumar et al 2012;Lee et al 2014). Day-7 blastocysts were frozen immediately by liquid nitrogen with small volume of DPBS and stored at À808C until RNA extraction.…”

“…In vitro maturation of oocytes Cumulus-oocyte complexes (COCs) were collected from prepubertal pig ovaries obtained from a local slaughterhouse and in vitro maturation (IVM) performed as previously described (Kumar et al 2007(Kumar et al , 2013Lee et al 2014). Briefly, COCs were aspirated from ovarian follicles of 3-5 mm in diameter using a 19-guage needle attached to a 10-mL syringe.…”