Construction of interferon regulatory factor 3 guided RNA eukaryotic expression vector

doi:10.3877/cma.j.issn.2095-5820.2022.04.005

Abstract

Abstract:

Objective

To investigate the molecular mechanism of interferon regulatory factor 3(IRF3) mediated transcription-independent apoptotic by constructing IRF3 gRNA eukaryotic expression vector.

Methods

IRF3's structure was analyzed and IRF3 gRNA was designed at CHOPCHOP, two pairs of primers were designed at ClaI and NheI site of pLenti-U6-gRNA-PGK-Neo expression vector. IRF3 gRNA expression module was amplified by gene splicing by overlap extension PCR (SOE PCR), and constructed in pGM-simple T clonal vector, and was subcloned in pLenti-U6-gRNA-PGK-Neo expression vector by dual digestion and IRF3 gRNA expression module was verified by bacteria PCR and Sanger sequencing.

Results

IRF3 gRNA was acquired between BH3 like domain and C terminal phosphorylation sites. IRF3 gRNA expression module was generated by SOE PCR. pGM-simple T-IRF3-gRNA clonal vector was constructed with IRF3 gRNA expression module by TA clone. pLenti-U6-IRF3-gRNA expression vector was constructed by dual digestion, and verified by Sanger sequencing.

Conclusions

The pLenti-U6-IRF3-gRNA expression vector was successfully constructed by SOE PCR, which laid a solid foundation on study of IRF3 mediated transcription-independent apoptotic mechanism.

Key words: Interferon regulatory factor 3, SOE PCR, CRISPR-Cas9, gRNA

Siyu Ju, Zhuoyan Hou, Xi Wang, Shengyuan Jiang, Jiaqi Li, Haifei Lyu, Wen Liu, Zhiyong Zhang. Construction of interferon regulatory factor 3 guided RNA eukaryotic expression vector[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2022, 10(04): 215-221.

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References 30

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引物名称	引物序列	碱基（bp）
引物A	5’-GGGTTCGAATTTATCGATCACGAGACTAGC-3’	30
引物B	5’-GGGCCATTTCTACCAAGGCCGGTGTTTCGTCCTTTCC-3’	37
引物C	5’-GGCCTTGGTAGAAATGGCCCGTTTTAGAGCTAGAAATAGCAAGTTAA-3’	47
引物D	5’-GGGGTACCGAATTCGCTAGCTCTCGA-3’	26

引物名称	引物序列	碱基（bp）
引物A	5’-GGGTTCGAATTTATCGATCACGAGACTAGC-3’	30
引物B	5’-GGGCCATTTCTACCAAGGCCGGTGTTTCGTCCTTTCC-3’	37
引物C	5’-GGCCTTGGTAGAAATGGCCCGTTTTAGAGCTAGAAATAGCAAGTTAA-3’	47
引物D	5’-GGGGTACCGAATTCGCTAGCTCTCGA-3’	26

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