Construction of p53^V149F Mutant Target Carrier and Cell Model

Purpose To construct p53 gene mutation model, providing cell model resources for studying the occurrence and progression of tumor induced by p53 gene mutation.

Methods According to the sequence of p53^V149F mutation site, CRISPR/Cas9 gene editing technology was used to design and synthesize the single-strand wizard sgRNA online, and construct CRISPR target vector. The recombinant vector was transfected into cells, positive cell populations were sorted by flow cytometry, and cell genome was extracted. Sanger sequencing was conducted to analyze the cleavage of target sites by Cas9 nuclease, and homologous repair template was constructed. The editing efficiency was further detected by T7EN1 assay. The recombinant vector and homologous template were transfected into fibroblasts by electrotransfection, and cell lines containing target mutations were selected by monoclonal selection, PCR and Sanger sequencing.

Results The CRISPR/Cas9 vector targeting p53 gene was successfully constructed. Five clone sites with target mutation were obtained, and the mutation rate was 10%. There were jacking peaks in the sequencing results, indicating that the genotypes were diverse. The sequencing results of one cell clone showed no heteropeak, and the genotype was identified by TA clone. The results showed that one allele had p53^V149F mutation, and the other allele had 240 bp deletion.

Conclusion The cell line containing p53^V149F is constructed successfully and the function of the gene is preliminarily verified. It lays a preliminary foundation for the further construction of the model of the simultaneous modification of two genes, providing available cell model resources for drug screening, and laying a foundation for the creation of miniature pig experimental animal models that simulate the genetic pattern and clinical manifestations of disease occurrence.