Journal of Shandong University (Health Sciences) ›› 2023, Vol. 61 ›› Issue (8): 1-9.doi: 10.6040/j.issn.1671-7554.0.2022.1302

• 基础医学 •    

Construction of endogenous epitope-tagged H1FX cell lines and the chromatin distribution mapping

XU Yaning1,2, ZHANG Xianglin1,2, LIU Xiaoyu1,2, GUO Haiyang1,2   

  1. 1. Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan 250033, Shandong, China;
    2. Tumor Marker Detection Engineering Laboratory of Shandong Province, Jinan 250033, Shandong, China
  • Published:2023-08-30

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Abstract: Objective To tag endogenous H1FX with epitope sequences for chromatin immunoprecipitation sequencing(ChIP-seq)using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)genome editing technology, and to investigate the genomic binding sites and distribution of H1FX in prostate cancer 22Rv1 cells. Methods Specific guide RNA(gRNA)was designed and inserted into the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 to construct the pX330-H1FX recombinant plasmid. Cas9 nuclease was guided to the position close to the H1FX stop codon for cleavage. With plasmid pFETCh_Donor containing the 3×FLAG epitope tag, self-cleaving peptide 2A and geneticin resistance gene sequence as the vector, the recombinant plasmid pFETCh_Donor-HOMO was constructed by adding homology arms(HOMO)that were homologous to the region on both sides of the double strand break. PX330-H1FX recombinant plasmid and pFETCh_Donor-HOMO recombinant plasmid were co-transfected into prostate cancer 22Rv1 cells. DNA was cleaved and epitope tags were integrated by homologous recombination repair. Cells were selected using medium containing geneticin after 48 hours. ChIP was performed on endogenous epitope-tagged H1FX-FLAG cell lines using FLAG antibody. ChIP-enriched DNA and input DNA libraries were constructed and sent for sequencing after the librarys were qualified. Subsequently, H1FX ChIP-seq data and gene expression data were integrated and analyzed. Results The results of Western blotting showed that the cell line only expressing H1FX-FLAG fusion protein was screened. The results of polymerase chain reaction showed that the FALG epitope tag was integrated into the correct position of the genome. Sequencing results showed that the insertion sequence and junction sequence were correct, and the H1FX-FLAG cell line tagged with endogenous epitopes was successfully constructed. The integration analysis of H1FX ChIP-seq data and gene expression data showed that the promoter regions of highly expressed genes were more likely to lack H1FX binding. Conclusion The endogenous epitope-tagged H1FX-FLAG cell lines were successfully constructed. The genomic binding sites and distribution of H1FX in 22Rv1 prostate cancer cells were preliminarily analyzed, which laid a foundation for further study on the role of H1FX in prostate cancer.

Key words: Clustered regularly interspaced short palindromic repeats, H1FX, Linker histone, Homologous recombination, Epigenomics, Prostate cancer

CLC Number: 

  • R737.25
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