Derivation And Pluripotency Analysis Of ES Cells From Pre-implantation Embryos Produced By Somatic Cell Nuclear Transfer (SCNT)

Embryonic stem(ES) cells are derived from the inner cell mass of blastocysts produced in vivo or by in vitro fertilization.ES cells are pluripotent and can differentiate into all the cell types of three germ layers.Recently,ES cells have been successfully derived from embryos produced by somatic cell nuclear transfer(SCNT) and it has been proposed important for therapeutic purpose and basic research in the future.In the present study,we derived pluripotent embryonic stem(ES) cells from murine blastocysts produced by somatic cell nuclear transfer(SCNT).The pluripotecy and differentiation potential of the ntES cells are investigated by RT-PCR,immunocytochemistry staining and chimera production.We have generated more 70 ntES cell lines from different strain of mice which include 129OG2F1 and BDF1 mice through SCNT.In the study,we found the efficiency to successfully derive ntES cells lines from cloned embryos varied with different genetic background of donor cells used in SCNT.The BDF1 mouse ntES derivation efficiency was 10.62%,OG2 (B6xCBA) ntES was 10.00%,129OG2F1(129xB6x CBA) ntES was 37.32%, B6OG2F1(B6xB6xCBA) ntES was 10.16%,and ZEG(ICR) was only 1.04%. Obviously,compared to other mouse strains,129OG2F1 genetic background has exhibited advantages in ntES cell derivation.These ntES cells appeared to maintain normal ES appearance in vitro.Six ntES cell lines from 129OG2F1 mouse and six BDF1 ntES cells lines were chosen to investigate the pluripotency. Our results indicated five of the twelve ntES cell lines tested exhibited similar pluripotency compared to normal ES cells.The genes encoded transcription factors which are essential for maintaining ES cells pluripotency,including Oct-4, Sox2,Nanog and Rex-1,were expressed in the ntES cells as tested by RT-PCR and immunocytochemistry staining.The differentiation potential of the ntES cells were further investigated by chimera production.Five of the twelve ntES cell lines exhibited the potential to differentiate into all three germ layers with one of them could even differentiate into germ cells.Our results indicated that the ntES cells established in the present study exhibited similar pluripotency and differentiation potential compared to normal fertilized embryo produced ES cells. These similarities of ntES cells and normal ES cells indicated that therapeutic cloning in mice could provide a reliable model for preclinical stem cell research. The ntES cells have been proposed important for regenerative medicine in the future,some degenerative diseases or genetic diseases might be completely corrected by the ntES cells generated by using the somatic cells from the specific patient as donor cells for SCNT.