Stable mESC Line Generation Using CRISPR

Flag-USP21 WT/SD/SA mESC stable cell line was generating using CRISPR-CAS methods62 (link). In brief, guide RNA (gRNA) expression vectors were constructed for pGS3-T7-gRNA. The sequence of gRNA (target to Rosa26) is 5′-TCGTGATCTGCAACTCCAG-3′. Flag-USP21 was ligated to the Sall and MluI site of Rosa26-puro-CAG donor vector. Then, we inserted the Flag-USP21 into the mouse Rosa26 safe harbour site via homologous recombination. An amount of 40 μg donor vector was electroporated into 0.3–0.4 million E14 cells along with 5 μg each of the Rosa26 gRNA and pCas9-GFP plasmid (Amaxa kit R, program A-24; Lonza). One week after electroporation, cells were replated into 150-mm dishes at clonal density. Individual colonies were subsequently selected under the fluorescence microscope and expanded in 24-well dishes. Analysis of Flag-USP21 stable cell line by western blotting.

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Jin J., Liu J., Chen C., Liu Z., Jiang C., Chu H., Pan W., Wang X., Zhang L., Li B., Jiang C., Ge X., Xie X, & Wang P. (2016). The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog. Nature Communications, 7, 13594.

Publication 2016

Amaxa kit R,

Cell line Cells Clonal Crispr Dishes Donor Electroporation Fluorescence microscope Homologous recombination Mesc Mouse Plasmid Rna expression Rna sequence Vector

Corresponding Organization :

Other organizations : East China Normal University, National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Tenth People's Hospital, Tongji University, Beijing Proteome Research Center, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences

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Variable analysis

independent variables

Guide RNA (gRNA) expression vectors constructed for pGS3-T7-gRNA
Flag-USP21 ligated to the Sall and MluI site of Rosa26-puro-CAG donor vector
Electroporation of 40 μg donor vector and 5 μg each of the Rosa26 gRNA and pCas9-GFP plasmid into 0.3–0.4 million E14 cells

dependent variables

Flag-USP21 stable cell line

control variables

Electroporation settings (Amaxa kit R, program A-24; Lonza)
Cell line used (E14 cells)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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