CnAβ基因对大鼠RBL-2H3细胞脱颗粒的影响

doi:10.11843/j.issn.0366-6964.2021.02.024

Abstract

Abstract: Allergy is a common disease in humans and farmed animals, and it develops quickly. To insight into the molecular mechanism of allergic disease, this study was focused on the function of calcineurin A beta (CnAβ) gene on RBL-2H3 cell degranulation. CRISPR/Cas9 gene editing technology was employed to construct CnAβ gene knockout RBL-2H3 cell line, and the proliferation and degranulation of RBL-2H3 cell effected by CnAβ gene were surveyed. The first exon of rat CnAβ gene was selected as a knockout target for the design and synthesis of three single guide RNAs (sgRNA), to construct the pX459-CnAβ-sgRNA knockout plasmid, and the constructed plasmid was transferred into RBL-2H3 cells by Lipofectamine 3000. After puromycin screening, the RBL-2H3 cell line with CnAβ gene deletion was obtained by PCR and DNA sequencing, and then used to detect the effect of CnAβ gene deletion on cell proliferation and degranulation. The results indicated that CnAβ gene knockout RBL-2H3 cell line was successfully constructed, and CnAβ deficiency had no significant effect on the proliferation, granule formation or granule content of RBL-2H3 cell, but significantly inhibited the degranulation of RBL-2H3 cells mediated by cell surface receptors. This research will contribute to an in-depth understanding of the mechanism of animal allergic diseases and provide a theoretical basis for the prevention of allergic disease.

Key words: mast cells, RBL-2H3 cells, CRISPR/Cas9, gene knockout, degranulation

CLC Number:

R593

LI Yanhong, LIU Jie, WU Zhengli. Effect of CnAβ Gene on Degranulation of Rat RBL-2H3 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 515-524.

References 33

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Effect of CnAβ Gene on Degranulation of Rat RBL-2H3 Cells

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