Effect of CRISPR/Cas9-mediated PD-1 gene knockout on proliferation, phenotype,IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys
10.3872/j.issn.1007-385x.2020.07.006
- VernacularTitle:CRISPR/Cas9介导的PD-1基因敲除对食蟹猴T细胞增殖、表型及IFN-γ
- Author:
MIAO Yi
1
,
2
;
DONG Jian
3
;
JIAO Deling
4
;
GAO Change
3
;
ZHANG Chao
1
,
2
Author Information
1. 1. Department of Oncology, Qujing First People'
2. s Hospital, Qujing 655000, Yunnan, China
3. 2. Key Laboratory of Cell Therapy Technology and Translational Medicine of Yunnan Province, the Third Hospital Affiliated to Kunming Medical University, Kunming 650118, Yunnan, China
4. 3. Key Laboratory of Conservation and Utilization of Biological Resources, Yunnan Agricultural University, Kunming 650201, Yunnan, China
- Publication Type:Journal Article
- Keywords:
programmed death receptor 1 (PD-1);
Cynomolgus monkey;
CRISPR/Cas9 gene editing technique;
gene knockout
- From:
Chinese Journal of Cancer Biotherapy
2020;27(7):757-763
- CountryChina
- Language:Chinese
-
Abstract:
[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation,
phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey
was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of
Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis
and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted
for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3
antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression
levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells
with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly
(both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.
- Full text:20200706.pdf
