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Expression of Nanog Gene in Hela Cells and the Effect of Its siRNA on Mouse Embryonic Stem Cells

Author: ZuoLin
Tutor: WangHuaYan;ZuoZhongYing
School: Northwest University of Science and Technology
Course: Clinical Veterinary Medicine
Keywords: Embryonic stem cells Nanog gene Expression vector RNA interference Small interfering RNA fragments
CLC: Q343
Type: Master's thesis
Year: 2007
Downloads: 153
Quote: 2
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Abstract


Embryonic stem cells from the the mammalian blastocyst inner cell mass, has the ability to self-renewal, unlimited proliferation, and has features to differentiate into various cell types. The study of the molecular mechanisms of embryonic stem cell pluripotency is a hot research topic in recent years. Nanog gene as an important factor for maintaining the pluripotency of ES cells and early embryonic cells, research Nanog mechanism of gene expression in ES cells and early embryonic cells as well as the relationship with LIF/STAT3 and Oct3 / 4 of pluripotent stem cells, ES cells, will help to understand the molecular mechanism to maintain pluripotency. The homeotic protein Nanog independent of the pathway LIF/STAT3 maintain the self-renewal capacity of embryonic stem cells. Nanog, Oct4 and Sox2 maintain embryonic pluripotent stem cells and the inner cell mass of the main regulatory factors. Nanog gene expression in embryonic stem cells or teratoma cells in the adult cells do not express. In mouse embryonic stem cells, using RNA interference inhibits Nanog gene expression will lead to embryonic stem cells to extraembryonic endoderm differentiation, while promoting extra-embryonic endoderm gene gata4 the expression of gata6 and lamininB1. The Nanog gene helps reveal an important way for embryonic stem cell pluripotency and self-renewal capacity and regulatory pathways induced embryonic stem cells differentiation is of great significance. The test clone a mouse Nanog gene construct eukaryotic expression vector of the Nanog gene Nanog gene was successfully expressed in Hela cells, and detection of Nanog gene Hela cell growth characteristics. Based on the Nanog gene sequences designed three pairs of siRNA interference against Nanog gene fragment and a pair of meaningless siRNA fragments as a negative control. Interference fragment transfected mouse ES cells and P19 cells observed interference Nanog gene expression on the growth and differentiation of stem cells. The specific content of the test as described below: 1. Primers were designed according to the the GeneBank login sequence, cloned mice Nanog cDNA contains 915bp sequence analysis of the cloned fragment, the Nanog gene the published mouse ES cells results GeneBank sequence (XM 1 32755, gi: 20831594) homology of 99.7%. The amplified gene fragment was inserted into the pEGFP-C1 vector construct Nanog gene eukaryotic expression vector pG-Nanog. Hela cells transfected recombinant vector, RT-PCR proved successful expression of Nanog gene in Hela cells. G418 to select stable cell transfection strains, by measuring cell growth curve, PCNA staining, cell cycle detection test, to prove that the the Nanog gene can promote the proliferation of Hela cells, prompting Hela cells into S phase. Synthesized according to the the Nanog gene sequence design three pairs of siRNA interference fragment N301, N749, N845, and a pair of meaningless siRNA fragment as a control. Using Lipofectamine 2000 these siRNA transfected ES-129 cells (from 129 mice) cultured after transfection for 24 h, to extract cells were collected using TriZol of total cellular RNA, semi-quantitative RT-PCR analysis screened the best jamming effect siRNA N301. N301 siRNA transfected ES-129 cells, cultured for 24h, 48h, ES cells sets off the start loose, but there are still a small number of cells to maintain pluripotency AP staining was weakly positive. P19 cells transfected with N301, cultured 24h to 48h, induce the formation of embryoid bodies decreased ability embryoid bodies formed low vitality, poor adherent.

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