Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. gRNAs and GFP Donor Template
2.3. Generation of Stable MSB-1 and HP8 Cell Lines Expressing Cas9
2.4. Generation and Characterization of pp38 Deletion Cell Line HP8-Δpp38
2.5. Reactivation of MDV from MSB-1-Δpp38
2.6. Western Blotting Analysis
2.7. Growth of HP8-Cas9-Δpp38 Cells
2.8. Single Cell Sorting and Flow Cytometry Analysis
2.9. q-PCR for GFP Copy Number
3. Results
3.1. Editing of pp38 in MSB-1 Cells
3.2. Editing of pp38 in HP8 Cell Line
3.3. Insertion of Marker Gene into MDV Genome in HP8 Cells by NHEJ Pathway
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Primer | Sequence (5′-3′) |
---|---|
NF | TTGGAATAGCCCCCTTCCCC |
NR | TTCGAAGCAGAACACGAAGGG |
CF | GATTCCACCTCCCCAGAATCC |
CR | CAGAGAATGCAACAATGCGT |
MF | ATGGTGAGCAAGGGCGA |
MR | CCGGTGGTGCAGATGAAC |
ovoF | CACTGCCACTGGGCTCTGT |
ovoR | GCAATGGCAATAAACCTCCAA |
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Zhang, Y.; Luo, J.; Tang, N.; Teng, M.; Reddy, V.R.A.P.; Moffat, K.; Shen, Z.; Nair, V.; Yao, Y. Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System. Viruses 2019, 11, 391. https://doi.org/10.3390/v11050391
Zhang Y, Luo J, Tang N, Teng M, Reddy VRAP, Moffat K, Shen Z, Nair V, Yao Y. Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System. Viruses. 2019; 11(5):391. https://doi.org/10.3390/v11050391
Chicago/Turabian StyleZhang, Yaoyao, Jun Luo, Na Tang, Man Teng, Vishwanatha R.A.P. Reddy, Katy Moffat, Zhiqiang Shen, Venugopal Nair, and Yongxiu Yao. 2019. "Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System" Viruses 11, no. 5: 391. https://doi.org/10.3390/v11050391
APA StyleZhang, Y., Luo, J., Tang, N., Teng, M., Reddy, V. R. A. P., Moffat, K., Shen, Z., Nair, V., & Yao, Y. (2019). Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System. Viruses, 11(5), 391. https://doi.org/10.3390/v11050391