PhiC31整合酶电穿孔鸡成纤维细胞诱发位点特异性基因盒交换

doi:10.11843/j.issn.0366-6964.2023.06.013

Abstract

Abstract: The present study aimed to evaluate whether the direct protein delivery of PhiC31 integrase through electroporation could induce the PhiC31-mediated recombination in chicken DF-1 cells. Here, prokaryotic expression and affinity chromatography purification was used to produce a small ubiquitin-related modifier (SUMO)-tagged His-PhiC31 fusion protein, and a SUMO proteinase was utilized to generate a natural PhiC31 protein by removing SUMO-His tag. The two types of PhiC31 protein were respectively incubated with pBCPB+ plasmid in tubes to induce intermolecular recombination. Subsequently, a landing pad (LP) plasmid harboring the attP^TT-DsRed2-attP^CT fragment was electroporated into the chicken DF-1 cells, followed by another round of electroporation of the natural PhiC31 protein along with a donor plasmid carrying the attB^TT-EGFP-HiBiT-attB^CT segment. The results showed that a prokaryotic expression vector (pET-28a-SUMO-PhiC31) was successfully constructed, and SUMO-His-PhiC31 fusion protein was expressed in a resolvable pattern in E.coli with induction of isopropyl-β-D-thiogalactoside (IPTG), and amount of the purified fusion protein reached 12 mg·L^-1; both SUMO-His-PhiC31 and natural SUMO-free PhiC31 were capable of triggering the intermolecular recombination between attP and attB sites of pBCPB+ plasmid, and the recombinant efficiency of SUMO-His-PhiC31 was comparable to that of PhiC31 proteins (50% vs. 52%); co-transfection of DF-1 cells with a PhiC31 expressing vector pCMVInt, LP and donor plasmids resulted in conversion of DsRed2 to EGFP, which was confirmed by fluorescent microscopy; the same results were observed when LP plasmid, the donor plasmid plus PhiC31 protein were successively electroporated to DF-1 cells, implying the occur of PhiC31-mediated recombination between attP^TT and attB^TT, attP^CT and attB^CT, respectively. The results indicated that the natural PhiC31 protein is biologically active. The protein is expected to serve as an important reagent in the Easi-CRISPR-TARGATT-mediated genome editing to generate transgenic chickens.

Key words: PhiC31 integrase, prokaryotic expression, electroporation, recombinase mediated cassette exchange (RMCE), DF-1 cells, chicken

CLC Number:

S831.2

WANG Hui, XU Mengyuan, LIU Lingkang, ZHENG Xibang, LI Gonghe, WU Wende. PhiC31 Integrase Triggers Site-specific Cassette Exchange in Chicken Fibroblast Cells through Electroporation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2330-2342.

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PhiC31 Integrase Triggers Site-specific Cassette Exchange in Chicken Fibroblast Cells through Electroporation

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