Expression of Imprinted Genes Kcnq1 and Cdkn1c During the Course of Differentiation from Mouse Embryonic Stem Cells into Islet-like Cells in vitro

Abstract

To study the effects of inducement on the expression of mouse embryonic stem cells SF1-G imprinted genes, Kcnq1 and Cdkn1c during the course of differentiation into islet-like cells in vitro. Mouse embryonic fibroblasts (MEFs) were isolated from pregnant mice embryos and fibroblast feeder cells were prepared by treating 3–5th generations MEFs with Mitomycin C. Moreover, mouse embryonic stem cells were induced to differentiate into islet-like cells directly. RT-PCR and Immunofluorescence staining were used to test the expression of islet cell-specific markers. Cells were collected at various stages throughout the differentiation process and the imprinted genes Kcnq1 and Cdkn1c were tested by reverse transcription-polymerase chain reaction fragment length polymorphism (RT-PCR/RFLP). In the present study, we found that cells appear islet cell-specific gene expression. Furthermore, immunofluorescence shows us that the islet cell-specific hormone protein can be measured at stage, which confirms that the embryonic stem cells can be successfully induced into islet-like cells in vitro. RT-PCR/RFLP analysis showsthat imprinted genes Kcnq1 and Cdkn1c are biallelic expression in the differentiated cells, suggestive of loss of imprinting (LOI), while these genes demonstrate maternal monoallelic expression in the undifferentiated cells’ continued subculture; this marks the maintenance of imprinting (MOI). Our data indicate that mouse embryonic stem cells are induced into islet-like cells in vitro. The gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells during the induction in vitro.

Key words

embryonic stem cells - islet-like cell - imprinted gene - epigenetics - differentiation

• References

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