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* These authors contributed equally
We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.
T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining naïve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate naïve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.
Th17 cells, a unique subset of CD4+ T helper cells, are vital for eradicating extracellular bacteria and fungi and play a significant role in various autoimmune diseases1,2,3. Emerging evidence suggests that Th17 cells exhibit heterogeneity, functioning in both pathogenic and non-pathogenic conditions, influenced by environmental and genetic factors. Elucidating the regulatory processes that control the differentiation of Th17 cells, plasticity, and heterogeneity is crucial for the advancement of more effective immunotherapeutic strategies.
Genetically modified animals have been widely used to unveil the key regulators of Th17 cell differentiation and functions. Using genetically modified animals involves complete manipulation in vivo, providing authenticity and systematic study of its role in physiological conditions or disease models. Nevertheless, high-throughput screening with this approach is largely impractical. In vitro polarization assays provide an alternative for studying Th17 cell differentiation. Interleukin 6 (IL-6) in combination with transforming growth factor β1 (TGFβ1) has been shown to promote the development of non-pathogenic Th17 cells, while IL-6, IL-1β, and IL-23 are implicated in driving the differentiation of pathogenic Th17 cells (pTh17)4,5.
The emergence of CRISPR/Cas9 technology has facilitated precise genome editing at specific bases. When combined with retroviral transduction, this approach provides a potent, efficient, and economical genetic method for screening and functionally studying potential regulators in Th17 cells6,7. In this study, we improved the procedure for retroviral transduction within Th17 polarization system. Using a retroviral system, we infected pre-established Cas9-expressing activated naïve T cells from mice. The cells were transduced with guide RNA (gRNA) constructs driven by the U6 promoter, along with genes encoding fluorescent reporter proteins under the control of the EF1a promoter to facilitate the knockout of the target gene. Then, the transduced T cells were cultured under specific cytokine conditions to induce differentiation into Th17 cells. Notably, knocking out RoRγt significantly reduced IL-17A production compared to the control group. The effectiveness of this system depends on optimized retrovirus production and transduction conditions for activated primary T cells, providing a rapid and practical approach for studying specific genes in Th17 differentiation and function.
All procedures were approved by the Experimental Animal Welfare Ethics Committee, Renji Hospital, Shanghai Jiao Tong University School of Medicine and are in compliance with institutional guidelines.
1. Retroviral production
2. Retroviral infection of activated CD4 + T cells and Th17 differentiation
3. Evaluation of transduction efficiency and differentiation results
In the study, we cloned the sgRNA target to Rorc and sgRNA-non-targeting coding sequences into pMX-U6-MCS vector with mCherry fluorescent protein (Figure 1A,B). Retrovirus production was carried out according to the protocol outlined in Figure 2. The transfection was initiated on day 0, and the retroviral harvest occurred on day 2. Transfection efficiency can be tested by the mCherry fluorescence intensi...
CRISPR/Cas9 genome editing via retroviral delivery is a robust method for exploring the roles of helper T cells. This protocol offers a rapid and effective approach to examining specific genes involved in Th17 differentiation and function. Several critical steps must be carefully followed to achieve optimal results. First, for enhanced gene knockout efficiency, gRNAs should be carefully selected. Given the risk of off-target effects in CRISPR/Cas9 gene editing, it is prudent to choose 2-3 gRNAs with high scores, as ident...
The authors state that there are no conflicts of interest.
We acknowledge Dou Liu, Dongliang Xu, and Pinpin Hou from the core facility of the Shanghai Immune Therapy Institute for their support in utilizing the instruments. This work was supported by National Natural Science Foundation of China Grants 31930038, U21A20199, 32100718, and 32350007(to Linrong Lu); 32100718 (to Xuexiao Jin); Innovative research team of high-level local universities in Shanghai SHSMU-ZLCX 20211600 (to Linrong Lu); Internal Incubation Program RJTJ24-QN-076(to Zejin Cui). Figure 2 was prepared with Figdraw.
Name | Company | Catalog Number | Comments |
0.5 M EDTA (pH 8.0) | Solarbio | E1170 | |
100 mm cell and tissue Culture Dish | BIOFIL | TCD010100 | |
1 M Hepes (Free Acid, sterile) | Solarbio | H1090 | |
24-well cell and tissue culture plate | NEST | 702002 | |
48-well cell and tissue culture plate | NEST | 748002 | |
Brefeldin A Solution (1,000x) | BioLegend | 420601 | |
Brilliant Violet 650 anti-mouse CD4 Antibody (RM4-5) | BioLegend | 100546 | |
CD3e Monoclonal Antibody (145-2C11), Functional Grade, eBioscience | Invitrogen | 16-0031-82 | |
CD44 Monoclonal Antibody (IM7), PE, eBioscience | Invitrogen | 12-0441-83 | |
CD62L (L-Selectin) Monoclonal Antibody (MEL-14), APC, eBioscience | Invitrogen | 17-0621-82 | |
Cell counter | Nexcelom Bioscience | Cellometer Auto 2000 | |
Cell Strainer (40 μm) | biosharp | BS-40-CS | |
Cell Strainer (70 μm) | biosharp | BS-70-CS | |
Centrifuge | eppendorf | 5425 R | |
Centrifuge | eppendorf | 5810 R | |
ChamQ SYBR Color qPCR Master Mix | Vazyme | Q411-02 | |
ClonExpress II One Step Cloning Kit | Vazyme | C112 | |
DH5α Competent Cells | Sangon Biotech | B528413 | |
Direct-zol RNA Miniprep | ZYMO RESEARCH | R2050 | |
DMEM Medium | BasalMedia | L110KJ | |
EasySep Mouse CD4+ T Cell Isolation Kit | STEMCELL | 19852 | |
eBioscience Fixable Viability Dye eFluor 660 | Invitrogen | 65-0864-18 | |
EndoFree Mini Plasmid Kit II | TIANGEN | DP118-02 | |
ExFect Transfection Reagent | Vazyme | T101-01 | |
Fetal Bovine Serum, Premium Plus | Gibco | A5669701 | |
FITC anti-mouse IL-17A Antibody (TC11-18H10.1) | BioLegend | 506907 | |
Formaldehyde solution | Macklin | F864792 | |
HiScript IV RT SuperMix for qPCR(+gDNA wiper) | Vazyme | R423-01 | |
Ionomycin | Beyotime | S1672 | |
Mitomycin C | Maokang Biotechnology | 7/7/1950 | |
Mouse GRCm38 | NCBI | RefSeq v.108.20200622 | |
OPTI-MEM Reduced Serum Medium | Gibco | 31985070 | reduced serum medium |
Pacific Blue anti-mouse CD4 Antibody (RM4-5) | BioLegend | 100531 | |
PE/Cyanine7 anti-mouse CD25 Antibody (PC61) | BioLegend | 102015 | |
PE/Cyanine7 anti-mouse CD4 Antibody (GK1.5) | BioLegend | 100422 | |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140122 | |
PMA/TPA | Beyotime | S1819 | |
R26-CAG-Cas9 mice | Shanghai Model Organisms Center | Cat. NO. NM-KI-00120 | |
Recombinant Human TGF-beta 1 (CHO-Expressed) Protein, CF | R&D Systems | 11409-BH | |
Recombinant Murine IL-6 | PeproTech | 216-16 | |
Research Cell Analyzer | BD Biosciences | BD LSRFortessa | |
Research Cell Sorter | SONY | MA900 | |
RPMI 1640 Medium | BasalMedia | L210KJ | |
SimpliAmp Thermal Cycler PCR System | Applied Biosystems | A24811 | |
Sodium pyruvate solution (100 mM) | Sigma-Aldrich | S8636 | |
Ultra-LEAF Purified anti-mouse CD28 Antibody (37.51) | BioLegend | 102121 | |
Ultra-LEAF Purified anti-mouse IFN-γ Antibody (XMG1.2) | BioLegend | 505847 | |
Ultra-LEAF Purified anti-mouse IL-4 Antibody (11B11) | BioLegend | 504135 | |
Ultra-LEAF Purified anti-mouse IL-12/IL-23 p40 Antibody (C17.8) | BioLegend | 505309 | |
β-Mercaptoethanol (50 mM) | Solarbio | M8211 |
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