Establishment of Interleukin-1 Receptor Type 1 Knockout Hela Strain by CRISP/Cas9 System

Hanayama M, Ishiyama Y, Sadamura M, Moriyama A, Imaoka S, Tsunoda M and Asano M

Published on: 2022-05-18

Abstract

The aim of the present study was to establish the interleukin-1 receptor type 1 (IL-1R1) knockout culture cell strain using the CRIPS/Cas 9 system. Human ovarian cancer-derived fibroblastic cell line HeLa was used for the experiments. For transfection, all-in-one CRISP plasmid encompassing Cas9 cDNA and single guide RNA (sgRNA) was transfected using Lipofectamin 3,000 reagent. The knockout strain was selected by incubating the cells in the presence of G418. The PCR products was amplified with the specific primers flanking to the expected gene alteration site using the genomic DNA obtained from the candidate cells. The amplified PCR products were gel purified and subjected to degeneration and reannealing reaction followed by T7 endonuclease digestion. Western blotting and immunofluorescence staining was conducted to confirm the IL-1R1 knockout in a protein level. To further confirm the specific knockout of IL-1R1 gene, the responsiveness against recombinant human IL-1α (rh IL-1α) was examined.

PCR amplification resulted in the production of 733 bp band. The band was digested to 510 bp and 233 bp fragments in candidate cells. The lack of IL-1R1 was confirmed by western blotting and immunofluorescence staining. HeLa cells is known to secrete IL-8 in response to rhIL-1α. Based on this, CR-R1-4 was stimulated with rhIL-1α, however, it did not induce IL-8 secretion. On the other hand, forced expression of IL-1R1 by transient transfection of IL-1R1 expression plasmid rescued the IL-1α responsiveness. The results indicated the successfull establishment of IL-1R1 KO (CR-R1-4) cell. The IL-1R1 KO cell line could be used for the further elucidation of functional properties of IL-1α and its receptors. he CRISP/Cas9 system is a useful method to establish the cell line with desired gene alteration.