Mechanism Of Imprinting Change Of Kcnq1, Cdkn1c Caused By Differentiation From Mouse ES Cells To Islet-like Cells In Vitro

Chapter one expression of imprinted genes Kcnql, Cdknlc in the course of mouse embryonic stem cells SF1-G induced to differentiate into islet-like cells in vitroObjectiveMouse embryonic stem cells SF1-G were induced to differentiate into islet-like cells in vitro. Epigenetic stability of cells at different stages was observed by testing the parental origin of imprinted gene Kcnq1 and Cdknlc.Methods 1. Mouse embryonic fibroblasts (MEFs) was isolated from pregnant mice embryos. Fibroblast feeder cells were prepared by treating 3-5th generations MEFs with Mitomycin C. Embryonic stem cell line SF1-G cells was expanded on feeder cells in vitro.2. Refering to a three-phase protocol from Shi, mouse embryonic stem cells were induced into islet-like cells directly. Immunof-luorescence staining and RT-PCR were used to detect the expression of islet cell-specific marker in differentiated cells.3.Cells were collected at various stages during differentiation process. Imprinting status of imprinted genes Kcnq1, Cdkn1c was tested by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR/RFLP)Results1. SF1-G cells can be cultured and proliferated maintaining the undifferentiated state on the feeder cells.2. RT-PCR results showed cells appeared islet cell-specific gene expression; immunofluorescence showed that islet cell-specific hormone protein can be measured at stage 3, confirmed that the embryonic stem cells can be successful inducted into islet-like cells in vitro.3. RT-PCR/RFLP analysis showed that imprinted genes Kcnq1, Cdknlc were biallelic expression in the differentiated cells, suggesting that they were loss of imprinting (LOI), while these genes were maternal monoallelic expression in the undifferentiated cells continued subculture, which marked the maintenance of imprinting (MOI).Conclusions1. Refering to Shi'protocol, mouse embryonic stem cells were induced into islet-like cells in vitro.2. Differentiation, not merely the process of cell culture can lead to abnormal expression of some imprinted genes, suggesting that epigenetic instability exist during the process of differentiation in vitro. ObjectiveTo explore the mechanism of expression of imprinted genes Kcnq1, Cdknlc in embryonic stem cells culture and differentiation process in vitro, we observed the differentially DNA-methylated region KvDMRl methylation status in SF1-G cells before and after differentiation, which is a part of the imprinting contol region(ICR) of imprinted genes Kcnql, Cdknlc, as well as the DNA methyltransferase levels in various stages of differentiation.Methods1. The methylation status of CpG sites of imprinting control region KvDMRl of imprinted genes kcnql, cdknlc in SF1-G cells before and after differentiation was detected with Bisulfite sequencing PCR.1) undifferentiated cells and inducing terminal differentiated cells were collected, extraction of genomic DNA, bisulfite treatment of DNA, with specific primers PCR amplification of the differentially DNA-methylated region KvDMR1,2) PCR product connected to the ampicillin resistance T Vector plasmid was transformed into Competent bacteria, the latter was evenly coated in the ampicillin resistance on agar plate culture. positive bacterial clones were picked the next day, the LB medium containing ampicillin screened positive bacteria,3) PCR was used to verify that the purpose fragment was existence in bacteria, positive bacteria was sent to Shanghai sangon biotechnology services company sequencing, the results were analyzed by software to show the methylation status in KvDMR1 before and after differentiation.2. Western blot was performed to detect methyltransferase Dmnt1 and Dmnt3b level at various stages of cells differentiation.Results1.We totally sequenced 23 CpG methylation sites of KvDMR1 region. Among 9 DNA sequencing results from embryonic stem cells before differentiation, there were 4 showed 1-23 CpG sites were methylation, the remaining 5 showed unmethylated, so KvDMR1 region showed all CpG methylation or no methylation in all, which is consistent with the characteristic of differentially DNA-methylated region(DMR). In differentiated cells,11 DNA sequencing results showed that 5 of them have methylation of all CpG sites,4 samples have methylation in the first 18-23 CpG sites, while the remaining CpG sites did not methylate, and the other 2 showed none of all CpG sites methylation, suggesting the methylation occurred in unmethylated KvDMR1.2. Western blot results showed that after the induction of cells differentiation from the second stage, DNA methyltransferase Dmnt1 level significantly increased, DNA methyltransferase Dmnt3b significantly higher in the third stage, while simultaneously subcultured undifferentiated cells have no significant change.Conclusions1. In the course of differentiation, the changes in the first 18-23CpG sites methylation status of differentially DNA-methylated region KvDMR1 in imprinting control region may relate to imprinted genes Kcnq1, Cdknlc loss of imprinting.2. In the course of differentiation, the increase of DNA methyltransferase level may mediate KvDMR1 methylation status changes, ultimately lead to changes in imprinted gene activity, as well as leading to loss of imprinting.