Establishment Of R6-MEF Cell Line Using Tale-dTF Targeting Xist Intron 1

X chromosome inactivation is an important event in the early embryonic development of female mammals,and the noncoding gene Xist is a key regulator of X chromosome inactivation through coating the X chromosome to be inactivated by its RNA.In the process of somatic cell reprogramming,the inactivated X chromosome becomes reactivation with the down-regulation of Xist.In ESC,pluripotency factors(such as NANOG,SOX2 and OCT4 etc.)bind to the intron 1 to stabilize the pluripotency state of ESC.However,it has been reported that knockout of the intron 1 region does not affect the Xist expression and the efficiency of induced pluripotent stem cell(iPSC)generation,while transcriptional-activator-like effectors based designer Transcription Factor(Tale-dTF)R6 targeting the first intron region of Xist significantly improved the reprogramming of mouse embryonic fibroblast(MEF)to iPSC.In this study,theimmortalized R6-MEF cell lines were successfully establishedusing the Tale-dTF R6,and the R6-MEF cell lines werecharacterizedby cell growth curve,immunofluorescence staining,real-time quantitative PCR,flow cytometry analysis and so on.Compared with wild-type MEF cell lines,the R6-MEF cell lines had some unique characteristics showed as follows:1.The mophorlogy of R6-MEF cell lines prepared by liposome transfection method were similar tothose of the low-generation wild type MEF cell lines,while the growth rate were significantly higher comparing tothe wild type MEF cell lines.The R6-MEF cell lines can be passaged to more than 50 generations with no obvious abnormalities.2.Real-time quantitative PCR results showed that the pluripotency genes Oct4,Sox2,Nanog and reproductive-related gene Prdm14 in R6-MEF were up-regulated to a certain degree.However,the immuno:fluorescence staining of NANOG in R6-MEF was negative.The results of immunofluorescence stainingindicated that R6 reduced the level of H3K27me3 in female R6-MEF.3.The Flow cytometry analysis showed that GOF-GFP in wild-type MEF cell lines was not activated,while GOF-GFP in R6-MEF cell lines was weakly positive.4.Karyotype analysis showed that R6-MEF cells and mouse fetal fibroblasts(MEF)chromosomes were 40,no significant abnormalities.In conclusion,the R6-MEF was a new type of cell lines between wild-type MEF and pluripotent stem cell(such as induced pluripotent stem cell),and the pluripotency of this cell line was far weaker than currentl y established pluripotent stem cells.Thus,the immortalized R6-MEF cell lineswere much closer to wild-type MEF cell lines,which provides new ideas for stem cell research and the establishment of animal breeding models.