Generating Xist Deletion Mutant ES Cells

16.7 female ES cells were used to generate Xist repeat E deletion mutant [30 (link)]. ES cells were grown under standard conditions as described except for the addition of 2i inhibitors (3 μM CHIR99021 and 1 μM PD0325901, LC laboratories) [28 (link)]. Xist expression from tet-inducible Xist cDNA transgene in T20 cell lines was induced by adding 1 μg/ml doxycycline (Dox) in the culture medium for differentiation for 2 days [21 (link)]. Cre-LoxP mediated insertion of the inducible Xist cDNA transgenes was done as described [21 (link)].

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Yue M., Ogawa A., Yamada N., Charles Richard J.L., Barski A, & Ogawa Y. (2017). Xist RNA repeat E is essential for ASH2L recruitment to the inactive X and regulates histone modifications and escape gene expression. PLoS Genetics, 13(7), e1006890.

Publication 2017

PD0325901

Cdna Cell lines Chir99021 Culture medium Deletion Doxycycline Es cells Female Grown Inhibitors Pd0325901 Transgenes

Corresponding Organization : Cincinnati Children's Hospital Medical Center

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Variable analysis

independent variables

Xist repeat E deletion mutation
Addition of 2i inhibitors (3 μM CHIR99021 and 1 μM PD0325901)
Induction of Xist expression from tet-inducible Xist cDNA transgene by adding 1 μg/ml doxycycline (Dox)

dependent variables

Xist expression

control variables

Standard growth conditions for ES cells

controls

Positive control: Xist expression from tet-inducible Xist cDNA transgene in T20 cell lines
Negative control: Not explicitly mentioned

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