Embryonic stem cells (ESC) are derived from the inner cell mass of blastocysts and proliferate extensively while maintaining pluripotency. They can be used for the treatment of juvenile diabetes, Parkinson’s disease, heart failure, and spinal cord injury. However, the use of embryos and tissue rejection remain concerns for ESC transplantation. Reprogramming of somatic cells may be done by different methods such as somatic cell nuclear transfer (Wilmut et al. 1997), fusion of somatic cells (Cowen et al. 2005), treatment with the extract of the pluripotent stem cells (Johnson Rajasingh 2008), and by the stable ectopic expression of defined factors in the somatic cells (Takahashi and Yamanaka 2006). Several transcription factors, including Oct3/4 (Nichols et al. 1998; Niwa et al. 2000), Sox2 (Avilion et al. 2003), and Nanog (Chambers et al. 2003; Mitsui et al. 2003), function in the maintenance of pluripotency in both early embryos and ESC. Takahashi and Yamanaka reported reprogramming the fibroblast cells into stem cells by introducing Oct3/4, Sox2, c-Myc, and Klf4 in mouse embryonic and adult fibroblasts. Yu et al. (2007) demonstrated that four transcription factors (OCT-4, SOX2, NANOG, and LIN28) are sufficient to reprogramme human somatic cells to pluripotent stem cells that exhibit the essential characteristics of ESC. Nakagawa et al. (2008) used three factors (OCT3/4, SOX2, and KLF4) for human iPS cell production from somatic cells. We are trying to reprogramme the adult goat fibroblast cells in induced pluripotent stem cells by using ectopic expression of transcription factors such as Oct-4, Sox2, Nanog, and Lin28. We collected the ovaries from a slaughtered animal from Delhi and collected the oocytes from ovaries. Then after the collection, A and B grade oocytes were selected. Selected oocytes were processed and incubated in in vitro maturation media for 24 h. We collected semen from a male goat, and it was processed and capacitated in sperm TALP. Capacitated sperms were used for IVF of the in vitro matured oocytes in ferTALP. After 12 h sperm were washed from oocytes in embryo developing media (EDM), and oocytes were cultured (in vitro) in EDM. After 24 h cleavage occurred. The cleaved embryos were cultured for 6 to 7 days. At the 7th day, we got blastocysts. From these blastocysts, inner cell mass was isolated enzymatically and cultured to get ESC. The ESC were cultured for 7 passages and used for RNA isolation. The RNA was isolated from these stem cells by the Trizol method. Complementary DNA was prepared by RT-PCR. Using gene-specific primer for Oct-4, Nanog, and Sox2, DNA was amplified. The DNA for the Oct-4, Nanog, and Sox2 genes was cloned in pJET cloning vector and transformed in Top10 E. coli competence cells. After screening, plasmid was isolated and sent for sequencing. Sequences were analysed and the complete open reading frame was created for Oct-4, Nanog, and Sox2.
An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells
