Expression and quantification of Oct-4 gene in blastocyst and embryonic stem cells derived from in vitro produced buffalo embryos

Manjinder Sharma, Rajesh Kumar, Pawan K Dubey, Om Prakash Verma, Amar Nath, G Saikumar, G Taru Sharma
In Vitro Cellular & Developmental Biology. Animal 2012, 48 (4): 229-35
The POU-domain transcription factor Pou5f1 (Oct-4) is involved in transcriptional regulation during early embryonic development and cell differentiation. Despite highly conserved genomic organization of Oct-4 gene in mammals, expression pattern of Oct-4 is highly variable in different species. In the present study, expression pattern of Oct-4 in buffalo blastocyst, trophoectoderm (TE), and embryonic stem cells (ESCs) was investigated. For the derivation and characterization of buffalo ESCs, inner cell masses (ICMs) were isolated from 18 hatched and 21 expanded in vitro produced buffalo blastocyst and cultured over mitomycin-C-treated buffalo fetal fibroblast feeder layer. Alkaline phosphatase (AP) activity, SSEA-1 and 4, TRA 1-60 and 1-81, and Oct-4 proteins were localized in ICM, TE, and ESCs. Quantification of Oct-4 was done by amplifying a transcript of 125 base pairs by real-time polymerase chain reaction. Primary cell colony formation was higher (P < 0.05) in hatched blastocyst (83.33%, 15/18) compared to mechanically isolated ICMs from expanded blastocyst (52.38%, 11/21). Undifferentiated buffalo ESCs were positive for AP and expressed Oct-4, SSEA-1 and 4, TRA-1-60, and TRA-1-81 proteins. Oct-4 transcripts and proteins were detected in the ICM, TE cells and were invariably present in ESCs; however, expression level of Oct-4 transcript were significantly higher in ICM and ESCs as compared to TE cells. In conclusion, expression of Oct-4 is not only restricted to the ICM and ESCs but its expression was also detected in TE cells suggesting that instead of using Oct-4 as a single marker, it is better to have other flanking molecular markers for the identification of buffalo pluripotent embryonic stem cells.

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