Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Ethics Statement
2.3. Embryo Manipulation and Genotyping of Pups Produced from Injected Embryos
2.4. Western Blotting
2.5. Histology and Immunohistochemistry
2.6. Flow Cytometry
2.7. Reverse Transcriptase Quantitative PCR (RT-qPCR)
2.8. In Vivo Infection of HAdV-C6 in Wild Type and RAG1-86nt Syrian Hamsters
2.9. Determining the Anti-Ad6 Neutralizing Antibody (NAb) Titers in the Serum
2.10. Statistical Analysis
3. Results
3.1. Genetic Targeting of the Non-Core Domain of Hamster RAG1 Protein
3.2. The 86-nt Deletion Abolishes the Expression of Full-Length RAG1 Protein
3.3. Off-Target Effect Analysis
3.4. RAG1-86nt Hamsters Are Atrophic in Lymphoid Organs
3.5. Naïve RAG1-86nt Hamsters Have Severely Reduced Size of T and B Lymphocyte Compartments
3.6. RAG1-86nt Hamsters Develop a Partial Adaptive Immune Response after Intranasal Infection with HAdV-C6
4. Discussion
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Category | Name | Sequence (5′-3′) | Note |
---|---|---|---|
For designated target | GH RAG1-F | AGACAAAGCAATTCACCAAG | For sgRNA/Cas9-RAG1 |
GH RAG1-R | ATCGGAGAATGCAGATTCTG | For sgRNA/Cas9-RAG1 | |
For potential off-site targets | OT1F | GGTTGACCTCTGGCCTAGAC | |
OT1R | CCTGTGCTTTGTTGGTTGTG | ||
OT2F | CCTATCCGCATTGTCCCATC | ||
OT2R | ATGGGGCTAATCTGGCGCAG | ||
For RT-qPCR | CD4-F | CATCGTAACCCAGAACCAGAAA | |
CD4-R | CCCTCGTATAGACTGTGGTAGAT | ||
CD8β-F | CCAGAATCCAGAACCACTCACA | ||
CD8β-R | CGGTCCAAGAAGGGTACAAGAA | ||
CD3ʏ-F | GAGCACAATCAGTCCCTACAA | ||
CD3ʏ-R | CTTTGCTCCTTGACACCAATAAG | ||
CD22-F | CTGCAAGAGGTCTTCCTGTATC | ||
CD22-R | GAAGACTCGATCCTGGTGTTATT | ||
IgM Receptor-F | ACCTCAGTTCAGCAAGCAATC | ||
IgM Receptor-R | CCTTGGTTCTCTCTCCCATACT | ||
CD94-F | CTCATCTCTAGTGTGCTTGGTG | ||
CD94-R | ATGGGACATGTTCTTTCAGGAG | ||
Klrg1-F | GAGGAATGGTAGCCACTGTTT | ||
Klrg1-R | TGTAAGGAGATGTGAGCCTTTG | ||
CD68-F | CCTGTCTCTCTCGTTTCCTTATG | ||
CD68-R | GTGGGAAGGACACGTTGTATT | ||
RPL18-F | GTTTATGAGTCGCACTAACCG | ||
RPL18-R | TGTTCTCTCGGCCAGGAA |
No. Inject Zygotes | No. Survived | No. Embryos (Pups) | No. Litters (Recipients) | No. Positive Pups (%) |
---|---|---|---|---|
119 | 103 | 87 (21) | 3 (3) | 6 (28.6) |
Off-Targets Sequence (5′-3′) | PAM | Off-Target Score | Sequence ID | Location of Sites | |
---|---|---|---|---|---|
OT1 | GGTTTTAGGGATCGATGTCA | AAG | 0.7 | NW_004801621.1 | 2321825 to 2321847 |
OT2 | GGTTTTAAGGATGGATGTGA | AGG | 0.7 | NW_004801607.1 | 1888496 to 18885018 |
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Miao, J.; Ying, B.; Li, R.; Tollefson, A.E.; Spencer, J.F.; Wold, W.S.M.; Song, S.-H.; Kong, I.-K.; Toth, K.; Wang, Y.; et al. Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9. Viruses 2018, 10, 243. https://doi.org/10.3390/v10050243
Miao J, Ying B, Li R, Tollefson AE, Spencer JF, Wold WSM, Song S-H, Kong I-K, Toth K, Wang Y, et al. Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9. Viruses. 2018; 10(5):243. https://doi.org/10.3390/v10050243
Chicago/Turabian StyleMiao, Jinxin, Baoling Ying, Rong Li, Ann E. Tollefson, Jacqueline F. Spencer, William S. M. Wold, Seok-Hwan Song, Il-Keun Kong, Karoly Toth, Yaohe Wang, and et al. 2018. "Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9" Viruses 10, no. 5: 243. https://doi.org/10.3390/v10050243