| 1. |
- Chen, Liangliang, et al.
(författare)
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User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9
- 2018
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Ingår i: STEM CELLS INTERNATIONAL. - : HINDAWI LTD. - 1687-966X .- 1687-9678.
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Tidskriftsartikel (refereegranskat)abstract
- Loss-of-function studies are critically important in gene functional analysis of model organisms and cells. However, conditional gene inactivation in diploid cells is difficult to achieve, as it involves laborious vector construction, multifold electroporation, and complicated genotyping. Here, a strategy is presented for generating biallelic conditional gene and DNA regulatory region knockouts in mouse embryonic stem cells by codelivery of CRISPR-Cas9 and short-homology-arm targeting vectors sequentially or simultaneously. Collectively, a simple and rapid method was presented to knock out any DNA element conditionally. This approach will facilitate the functional studies of essential genes and regulatory regions during development.
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| 2. |
- Zhang, Wensheng, et al.
(författare)
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The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation
- 2019
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Ingår i: Cell Stem Cell. - : CELL PRESS. - 1934-5909 .- 1875-9777. ; 24:1, s. 138-
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Tidskriftsartikel (refereegranskat)abstract
- BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.
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