Title

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

Authors

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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