(piPSCs) or exposure to the oocyte cytoplasm after nuclear transfer can produce nuclear-derived ESCs Transfer (piPSCs-ntESCs). nuclear transfer reconstructed embryos with fluorescent markers piPSCs have ZsGreen for exogenous expression of Nanog and Lin28. reconstructed oocytes developed to morphologically normal 8-cell / morulae (35/93, 37.6%) and blastocyst (12/93, 12.9%).
Although most blastocysts green fluorescent protein-positive demonstrate efficient outcomes (8/10, 80%), do not form colonies and all cultures degenerate primer. Conversely, 15% of positive fluorescent 8-cell embryo stage / morula shows the results (6/40), with three primary colonies formed (7.5%). Third expanded and maintained as a line-ntESC piPSC.
These cell lines expressed the stem cell marker genes and proteins. subcellular fractionation protocol Although inactivation of one X chromosome, all lines piPSC-ntESC derivative form teratomas consisting of three embryonic germ layers. SSEA1 strong, 3, and 4 expression was detected in an 8-cell stage / morula embryos reconstructed from both piPSCs and pig embryo fibroblast (PEFs). SSEA3 is notably absent from the control IVF embryo pre-implantation stage.
Finally, we tried to establish ntESCs of 8-cell / morulae PEFs reconstructed using culture conditions similar to those for piPSC-ntESC derivation. Although eight colonies mainly derived from 107 embryos (7.5%), they all drop after the first part of culture.
Early and sustained expression of the reprogramming of key regulatory factors may be important for the derivation of pluripotent stem cells to derive piPSC-ntESCs of the 8-cell stage / morula, whereas SSEAs expression may be involved in stem cell colony formation initial phase.
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