Materials and Methods

No apparent p53 activation in CRISPR‐engineered gene‐edited rabbits

2. MATERIALS AND METHODS2.1. Animal and ethics statementThe gene‐editing rabbits were generated in our laboratory; the DMD, XIST, DMP1, FAM83h, FBN1 and CD300LF gene editing rabbits were generated by CRISPR/Cas9 system; the TYR, FUT1 and OTC gene editing rabbits were generated by BEs system. All protocols using rabbits were approved and performed with the guidelines of the Animal Care and Use Committee of Jilin University.2.2. Genotyping of the gene‐editing rabbitsGenomic DNA was extracted from a small piece of ear tissue of CRISPR‐edited rabbits using a TIANamp Genomic DNA Kit (Tiangen) according to the manufacturer's instructions. The PCR amplification primers are listed in Table S1.2.3. Western blotting assayWestern blotting was performed based on the previously described protocol. 6 Briefly, tissues were lysed with RIPA buffer supplemented with protease inhibitor (PI, Thermo Scientific) and phenylmethanesulfonyl fluoride (PMSF, Roche Applied Science) on ice for 30 min and vortex briefly evert 10 min. Protein concentrations were measured with BCA Protein Assay Kit (Beyotime). First of all, a total of 40 ug of protein from each tissue sample was separated by 12% SDS‐PAGE and then electrophoretically transferred to a polyvinylidene difluoride membrane (PVDF). Next, blocked the membranes with 3% BSA/Tris‐buffered saline/Tween (TBS‐T) for 1 h at room temperature, then incubated with anti‐p53 monoclonal antibody (1:2000, Proteintech 60283–2) and anti‐Beta actin monoclonal antibody (1:5000, Proteintech 60008–1) overnight at 4°C, and incubated with HRP‐anti‐mouse for 60 min at room temperature. Finally, bands were visualized by enhanced chemiluminescence solution (ECL, Meilun). Image‐J was used to quantify the band signals.2.4. Immunofluorescence stainingAn immunofluorescence assay was performed as previously described. 7 The paraffin‐embedded tissues were deparaffinized and antigen retrieval was performed using microwave oven heating. After blocking with 10% goat serum in PBS for 1 h at room temperature and incubated with an antibody against p53 (diluted in PBS with 1% BSA) at 4°C overnight (60283–2, Proteintech) was used. Then, the tissues were incubated for 1 h at room temperature with secondary antibodies (Alexa Fluor 555‐conjugated goat anti‐mouse IgG, Cell Signaling Technology). Finally, the nuclei were stained with 4’,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich). The coverslips were then sealed with glycerol and images were captured with a laser confocal microscopy (Fluoview FV1200, Olympus). Image‐Pro Plus software was used (Media Cybernetics, Rockville MD) to quantify the immunofluorescent signals.2.5. Statistical analysisData were statistically analysed with GraphPad Prism software (t‐test) and expressed as mean ±standard error of mean (SEM), and p <  0.05 was considered statistically significant.

Article TitleNo apparent p53 activation in CRISPR‐engineered gene‐edited rabbits

Abstract

The data that support the findings of this study are available from the corresponding author upon reasonable request.


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