The Construction Of PIKX-EGFP-C1Plasimd And Its Influnce On X-linked Genes In Clone Embryo

X-chromosome inactivation (XCI) refers to the silencing of one of the two Xchromosomes in females during early development, thus ensuring dosage compensation forX-linked gene products between XX females and XY males. Cloning mammals by somaticcell nuclear transfer (SCNT) is highly inefficient. Reprogramming errors appear to ariserandomly, but the nature of nonrandom, SCNT-specific errors also exists. Xist, a noncodingRNA that inactivates one of the two X chromosomes in females, was ectopically expressedfrom the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xiston Xa showed normal global gene expression and resulted in about an eight-to ninefoldincrease in cloning efficiency. My thesis involves integration of inducible RNAi element intobovine genome, and then conduct induction of shRNA in the eight cell stage of embryo, thuslaying foundations for the research on the ectopic X inactivation in cloned embryo. Results:1. We screened the most efficient shRNA by QRT-PCR by transfection and RNAi andthen ligased it to the pIKX-EGFP-C1plasmid. The Plamid were transformed into the E.coli(DH5α) and then sequenced. We chosoed the right ones for the next experiment.2. The pasmids pIKX-EGFP-C1and CMV-Int were co-transfected with X-treme GENEHP DNA Transfection Reagent into bovine fibroblast, and the cells were screened by G418.We harvested the single clones expressing Green Fluorescent Protein. The single clones weredetected by PCR and southern blot to find the positive single clones.3. The positive cells were conducted by SCNT, and the indusor was added on day3. Xistwas efficiently knockdowned. The ectopic expression of X-linked genes were rescued in malecloned embryo, while some of X-linked genes were rescued too far in female cloned embryo.