Inactivation of Klf5 by zinc finger nuclease downregulates expression of pluripotent genes and attenuates colony formation in embryonic stem cells

    Authors

    X. L. Long;D. K. Singla

    Comments

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    Abbreviated Journal Title

    Mol. Cell. Biochem.

    Keywords

    Self-renewal; Differentiation; Zinc finger nucleases; Klf5; ES cells; KRUPPEL-LIKE FACTORS; TRANSCRIPTION FACTORS; SELF-RENEWAL; RNA; INTERFERENCE; MAMMALIAN-CELLS; ES CELLS; MAINTENANCE; THERAPY; FAMILY; Cell Biology

    Abstract

    Recent studies suggest that Klf5 is required to maintain embryonic stem (ES) cells in an undifferentiated state. However, whether Klf5 can be inactivated by novel fusion technology of zinc finger nucleases (ZFN) has never before been examined. Therefore, we used ZFN technology to target the Klf5 gene in mouse ES cells, and examined the effects of the Klf5 gene on the expression of pluripotency-related genes, Oct3/4, Nanog, and Sox2 and on the self-renewal of ES cells. In Klf5-ZFN-transfected cells, expression of the Klf5 mRNA was downregulated by similar to 80 % compared to the control. Furthermore, expression of the Oct3/4 and Nanog mRNAs was significantly decreased in the Klf5-ZFN-targeted cells. RT-PCR analysis, however, showed no significant change in the level of Sox2 mRNA, but a decreased trend was evident in the Klf5-ZFN-targeted cells. Moreover, we observed the spontaneous differentiation of Klf5-ZFN-transfected cells and quantitative analysis revealed a significant decrease in colony formation in Klf5-ZFN-transfected cells. In conclusion, our data suggest that ZFN methodology is an effective approach to target the Klf5 gene and that Klf5 plays an important role in the maintenance of ES cell self-renewal.

    Journal Title

    Molecular and Cellular Biochemistry

    Volume

    382

    Issue/Number

    1-2

    Publication Date

    1-1-2013

    Document Type

    Article

    Language

    English

    First Page

    113

    Last Page

    119

    WOS Identifier

    WOS:000324253900011

    ISSN

    0300-8177

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