甲基结合蛋白1对小鼠胚胎干细胞增殖及克隆形态的影响
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摘要
目的:研究甲基结合蛋白1(Mbd1)对小鼠胚胎干细胞克隆形态及增殖的影响。方法:设计针对Mbd1转录起始位点的gRNA,将表达有gRNA和Cas9蛋白的质粒使用脂质体转染方法转入小鼠胚胎干细胞(J1);使用抗生素将未转染成功的细胞杀死后,细胞继续培养7 d,挑取单克隆细胞,采用PCR法筛选敲除Mbd1的纯合子细胞,观察Mbd1缺失对小鼠胚胎干细胞克隆形态的影响;使用细胞计数法检验Mbd1对胚胎干细胞增殖的影响。结果:通过Cripsr/Cas9成功获得敲除Mbd1的胚胎干细胞,Mbd1缺失后的小鼠胚胎干细胞丧失了常规小鼠胚胎干细胞的3D克隆形态,细胞呈现单层扁平克隆;Mbd1缺失后小鼠胚胎干细胞增殖速率变慢。结论:Mbd1通过影响小鼠胚胎干细胞克隆形态和增殖而影响胚胎干细胞的多能性。
Objective: To investigate the effect of methyl-CpG binding domain protein 1(Mbd1) on the colony morphology and proliferation of mouse embryonic stem cells(mESCs). Methods: A gRNA was designed to specifically target to the transcriptional start site of Mbd1 and cloned into pX459 plasmid, and then co-transfected with the plasmid which contains Cas9 gene into mESCs. mESCs expressing gRNA were selected by puromycin treatment, cultured for 7 days to form a colony and then the Mbd1 deletion was verified by genotyping using PCR. The efficiency of Mbd1 deletion was further confirmed using Western Blot. The morphology of mESCs was observed and cell proliferation was measured using cell counting method. Results: The expression of Mbd1 was completely abrogated by Cripsr/Cas9. The 3 D-colony morphology of mESCs was lost in Mbd1 knockout mESCs. The proliferation of Mbd1 knockout cells was slower than the wildtype cells. Conclusion: Mbd1 is important for the pluripotency maintenance of mESCs via regulating the colony formation and cell proliferation.
Objective: To investigate the effect of methyl-CpG binding domain protein 1(Mbd1) on the colony morphology and proliferation of mouse embryonic stem cells(mESCs). Methods: A gRNA was designed to specifically target to the transcriptional start site of Mbd1 and cloned into pX459 plasmid, and then co-transfected with the plasmid which contains Cas9 gene into mESCs. mESCs expressing gRNA were selected by puromycin treatment, cultured for 7 days to form a colony and then the Mbd1 deletion was verified by genotyping using PCR. The efficiency of Mbd1 deletion was further confirmed using Western Blot. The morphology of mESCs was observed and cell proliferation was measured using cell counting method. Results: The expression of Mbd1 was completely abrogated by Cripsr/Cas9. The 3 D-colony morphology of mESCs was lost in Mbd1 knockout mESCs. The proliferation of Mbd1 knockout cells was slower than the wildtype cells. Conclusion: Mbd1 is important for the pluripotency maintenance of mESCs via regulating the colony formation and cell proliferation.
引文
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[5] SHIBA Y,FERNANDES S,ZHU W Z,et al.Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts[J].Nature,2012,489 (7514):322-325.
[6] KRIKS S,SHIM J W,PIAO J,et al.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease[J].Nature,2011,480 (7378):547-551.
[7] ZHU D,FANG J,LI Y,et al.Mbd3,a component of NuRD/Mi-2 complex,helps maintain pluripotency of mouse embryonic stem cells by repressing trophectoderm differentiation[J].PLoS One,2009,4 (11):e7684.
[8] DOS SANTOS R L,TOSTI L,RADZISHEUSKAYA A,et al.MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner[J].Cell Stem Cell,2014,15 (3):102-110.
[9] RAIS Y,ZVIRAN A,GEULA S,et al.Deterministic direct reprogramming of somatic cells to pluripotency.[J].Nature,2013,502 (7469):65-70.
[10]LU Y,LOH YH,LI H,et al.Alternative splicing of MBD2 supports self-renewal in human pluripotent stem cells[J].Cell Stem Cell,2014,15 (1):92-101.
[11]LI X,BARKHO B Z,LUO Y,et al.Epigenetic regulation of the stem cell mitogen Fgf-2 by Mbd1 in adult neural stem/progenitor cells[J].J Biol Chem,2008,283 (41):27644-27652.
[12]ZHAO X,UEBA T,CHRISTIE B R,et al.Mice lacking methyl-CpG binding protein 1 have deficits in adult neurogenesis and hippocampal function[J].Proc Natl Acad Sci U S A,2003,100 (11):6777-6782.
[13]ZHANG P,RAUSCH C,HASTERT F D,et al.Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner[J].Nucleic Acids Res,2017,45 (12):7118-7136.
[14]NEMUDRYI A A,VALETDINOVA K R,MEDVEDEV S P,et al.TALEN and CRISPR/Cas Genome Editing Systems:Tools of Discovery[J].Acta Naturae,2014,6 (3):19-40.
[15]WANG H,LA RUSSA M,QI L S.CRISPR/Cas9 in Genome Editing and Beyond[J].Annu Rev Biochem,2016,85:227-264.
[16]HRYHOROWICZ M,LIPI■,et al.CRISPR/Cas9 immune system as a tool for genome engineering[J].Arch Immunol Ther Exp,2017,65(3):233-240.
[17]JIANG F,DOUDNA J A.CRISPR-Cas9 structures and mechanisms[J].Annu Rev Biophys,2017,46:505-529.
[18] FUJITA N,TAKEBAYASHI S,OKUMURA K,et al.Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms[J].Mol Cell Biol,1999,19 (9):6415-6426.
[19]FUJITA N,SHIMOTAKE N,OHKI I.Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1 mechanism of transcriptional regulation by methyl-CpG binding protein MBD1[J].2000,20 (14):5107-5118.
[20]J?RGENSEN H F,BEN-PORATH I,BIRD A P.Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains[J].Mol Cell Biol,2004,24 (8):3387-3395.
[21]DOBLE B W,WOODGETT J R.GSK-3:tricks of the trade for a multi-tasking kinase[J].J Cell Sci,2003,116 (7):1175-1186.
[22]GINIS I,LUO Y,MIURA T,et al.Differences between human and mouse embryonic stem cells[J].Dev Biol,2004,15;269 (2):360-80.
[23]WHITE J,DALTON S.Cell cycle control of embryonic stem cells[J].Stem Cell Rev.2005,1 (2):131-138.