GENE EXPRESSION IN SCNT EMBRYOS AND THEIR DEVELOPMENTAL POTENCY IN SURROGATE GOATS
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Date
2014
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Nanaji Deshmukh Veterinary Science University, Jabalpur (M.P.)
Abstract
The somatic cell nuclear transfer (SCNT) technology has opened an opportunity to multiply genotypes of high genetic value, transgenesis, embryonic stem cell application, preservation of endangered species, pharmaceutical protein production etc. In spite of the widespread applications, the efficiency of SCNT is less in terms of viable offspring. Reprogramming failures of donor somatic cell and aberrant gene expression are considered as one of the important factor for less developmental potency of cloned embryos. In the present study, gene expression pattern of pluripotent marker genes in donor cells and SCNT embryos were evaluated in order to assess the extent of reprogramming. To increase the reprogramming efficiency of somatic cells as a nuclear donor, fibroblast cells were induced to reprogram by various methods viz., serum starvation, overconfluent cell line, roscovitin and aphidicloin treatment. The reprogrammed cell lines were analyzed for expression of pluripotent marker genes by real time RT-PCR. The reults showed that Oct-4, Nanog and Dnmt-1 genes were expressed in each of the reprogrammed cell lines at varying degree. Thus the findings suggested that the epigenetic markers had been erased during the process of reprogramming. SCNT embryos were produced by modified hand-made cloning technology using innovative micro tools under three different culture media. Parthenogenetic and in vitro fertilization (IVF) embryos were taken as control for assessment of developmental potential and transcription analysis, respectively. In the present study, ejaculated as well as epididymal sperms of buck were successfully used for fertilization of goat oocytes. In the experiment, RVCL-BLAST media showed highest developmental rate as compared to mSOF and RVCL. The transcriptional level of pluripotent marker genes (Oct-4, Nanog and Klf-4), growth promoting factors (IGF-2 and IGF-2R) and DNA methyltransferase (Dnmt-1) in SCNT embryos at 8-16 cells, morula and blastocyst stages was expressed differentially as compared to respective stages of IVF embryos. IVF and SCNT embryos were further transferred at 4-8 cells stages into fallopian tube of synchronized goats. Prior to embryo transfer, the presence of corpus luteum of synchronized goat were confirmed by laparoscopic examination. In a total attempt of 16 and 13 experimental trials for IVF and SCNT embryos respectively, one recipient each from both group was found to have vesicle like structure on ultrasonography during day 35-40. However, the same goats were declared as negative for pregnancy on three months and exhibited the sign of oestrous.