Construction of THP-1 cell line with GPR108 gene deletion by CRISPR / Cas9 system and exploration of its function
10.19405/j.cnki.issn1000-1492.2023.04.002
- Author:
Wenwen Wang
1
;
Zhenzhen Tu
1
;
Dandan Zang
2
;
Jing Wang
1
;
Yintao Zhang
1
;
Haisheng Zhou
3
Author Information
1. Dept of Biochemistry and Molecular Biology，Anhui Medical University，Hefei 230032
2. Center for Scientific Research，Anhui Medical University，Hefei 230032
3. Dept of Biochemistry and Molecular Biology， Center for Scientific Research，Institute of Dermatology，Anhui Medical University，Hefei 230032
- Publication Type:Journal Article
- Keywords:
GPR108;
THP-1;
CRISPR / Cas9;
IL-8
- From:
Acta Universitatis Medicinalis Anhui
2023;58(4):528-533
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a Tohoku hospital pediatrics-1 (THP-1) cell line with G protein-coupled recep- tor108 ( GPR108) deletion and explore its functions．
Methods:According to the requirements of the clustered regularly interspaced short palindromic repeats ( CRISPR) / CRISPR-associated protein 9 ( Cas9 ) system ，two single guide RNAs (sgRNA1 and sgRNA2) paring to the flanking fragments of human GPR108 gene were designed and synthesized．The two oligonucleotides were inserted in the pL-CRISPR. EFS．GFP vector to generate the new recombinant vectors ( pL-CRISPR. EFS．GFP-sgRNA1 and pL-CRISPR. EFS．GFP-sgRNA2 ) ．The recombinant vectors and packaging plasmids (pMD2. G and psPAX2) ，were co-transfected into 293T cells to generate virus for infecting THP-1 cells．The GFP + cells were screened and isolated in 96-well culture plates by flow cytometry to obtain single-cell clones．PCR and Western blot were used to detect whether GPR108 was successfully knocked out in THP- 1 cells．Both GPR108 + / + and GPR108 －/ － THP-1 cells were treated with lipopolysaccharide (LPS) ．Interleukin 8 (IL-8) derived from the THP-1 cells，which were treated by LPS，was detected with Western blot and cytometric bead array ( CBA) analysis.
Results:The recombinant lentiviral vector pL-CRISPR. EFS．GFP-sgRNA was successfully constructed and single-cell clone F9 was obtained by flow cytometric sorting after transfection of THP-1 cells．PCR and Western blot both confirmed that F9 was a GPR108 －/ － THP-1 single-cell clone． LPS stimulated GPR108 －/ － and GPR108 + / + THP-1 cells，both Western blot and CBA results showed a significant decrease in IL- 8 synthesis and secretion in GPR108 －/ － THP-1 cells．
Conclusion :The GPR108 －/ － THP-1 cell clone is success- fully obtained based on the CRISPR / Cas9 system．GPR108 deletion in THP-1 cells treated by LPS leads to a decrease of IL-8 expression and secretion．It lays the foundation for further research on the molecular mechanisms of GPR108 in the immune inflammatory response.
- Full text:2024071709554005736基于CRISPR_Cas9...-1细胞株及其功能初步研究_王雯雯.pdf
