Study Of The Effect Of Exogenous Transcription Factors Integration Sites On Ipscs Reprogramming

Induced pluripotent stem cells(iPSCs)were first generated from mouse fibroblasts by forced expression of four transcription factors Oct3/4,Sox2,c-Myc and Klf4 introduced by retroviruses vectors in 2006.iPSCs have the similar morphology and gene expression profiles with Embryonic Stem(ES)cells and also have the capability of self-renew and the potential to differentiate into other tissues and organs.It can be used for study embryo development,differentiation and drug screening.However,there are still some problems in iPSCs' application.One is the integration of exogenous transcription factors which may affect the pluripotency and safety of iPSCs.The retroviruses vectors used for induction can integrate into the target genome DNA randomly.Such random integrations may lead to iPSCs pluripotency defects by inactivate some functional genes,and can also cause tumorigenesis by activate related pro-oncogene.In this research,the integration sites of four exogenous transcription factors in three iPSCs that have been proved to possess the truly pluripotency by tetraploid complementation experiment were identified.Subsequently,the function and expression level of flanking genes were analyzed by bioinformatics.The potential correlation between the transcription factors integration sites and the pluripotency and safety in the three iPSCs was also investigated.Twenty-two integration sites of exogenous transcription factors were identified,thirty-nine flanking genes were involved.Seventeen(77.3%)integration sites were located in intergenic regions.GO analysis result showed,none of twenty-two flanking genes have correlation with oncogene or important genes in development and differentiation.About twenty-eight(71.8%)flanking genes were found have the function like the housekeeping gene and expression microarray data show that these flanking genes have no significant difference in expression level among the three iPSCs,MEF and ESCs,implying that the exogenous transcription factors integration have no significantly influence in the expression level of flanking genes.The integration sites of exogenous transcription factors were found to be located in the intergenic regions and in introns far from the transcription start site.There does not appear to have an effect on the flanking gene expressions.More,many of the flanking geen were found to be housekeeping genes,suggesting a friendly environment for the expression of exogenous transcription factors for iPSC induction.The developmental capacities of the iPS mice generated from the three iPSCs previously identified to have high pluripotency and ES mice were highly similar,but the F0 mice from these iPSCs were found to be tumorigenic.There were no protoongogene identified wihtin 500 KB of the excogenous transcription factor integration sites,but high expression level of c-Myc were noted,suggesting a contributing factor for the tumor formation in the relevant iPS mice.Thus,our study suggested that the integration sites of exonegeous genes may not affect the pluripotency of the resulting iPSCs,so much as the level of exonegeous genes expression.