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Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos.

ABSTRACT: To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos.Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos.A significantly (P?

SUBMITTER: Em S

PROVIDER: S-EPMC4389932 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Project description:The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical "S" shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10-15 and 8-12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

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Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos.

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