Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells
Abstract
:1. Introduction
2. Results
2.1. bTERT Is a Potent Factor for Immortalization
2.2. HMEJ-Based Site-Specific bTERT Insertion Enables Efficient Immortalization of Bovine Cells
2.3. Characteristics of Immortalized Bovine Cells Established via HMEJ-Based bTERT Integration
2.4. bTERT-Driven Immortalization via the CRISPR/Cas9 System Inhibited by the Post-Translational Modification in bBMMs
3. Discussion
4. Materials and Methods
4.1. Cell Isolation and Culture
4.2. Construction of Vectors
4.3. Cell Transfection
4.4. Reverse Transcription PCR and Quantitative RT-PCR
4.5. Western Blot
4.6. Establishment of Immortalized Bovine BFFs and bBMMs Using the HMEJ-Based CRISPR/Cas9-Mediated bTERT Expression at ROSA26 Locus
4.7. RNA Binding Protein Immunoprecipitation Assay
4.8. Telomerase Activity Assay
4.9. Relative Telomere Length Measurement by Quantitative Real-Time PCR
4.10. EdU(5′-ethynyl-2′-deoxyuridine) Incorporation Assay
4.11. Detection of Individual Colonies by PCR
4.12. Off-Target Analyses
4.13. Soft Agar Assay for Colony Formation
4.14. Karyotype Analysis
4.15. Senescence-Associated β-Galactosidase Staining
4.16. Cellular Proliferation Assay
4.17. Cell Cycle Analysis
4.18. FACS Analyses
4.19. Immunofluorescence Staining
4.20. Preparation of Cell Lysates
4.21. Co-Immunoprecipitation
4.22. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Construct | Enzymatic Activity | Telomere Length |
---|---|---|
bTERT | ++++ | ++++ |
hTERT | +++ | +++ |
mTERT | − | − |
bTERT-△C | ++ | − |
hTERT-△C | − | − |
mTERT-△C | − | − |
HHB | +++ | +++ |
MMB | ++ | + |
BBH | + | − |
MMH | + | − |
BBM | − | − |
HHM | − | − |
Immortalization Strategy | Passage 0 | Passage 5 | Passage 15 | Passage 35 | Passage 50 | Passage 80 | |
---|---|---|---|---|---|---|---|
Transient transfection system | hTERT | 26 | 1 | 0 | 0 | 0 | 0 |
25 | 0 | 0 | 0 | 0 | 0 | ||
16 | 0 | 0 | 0 | 0 | 0 | ||
bTERT | 25 | 2 | 1 | 0 | 0 | 0 | |
21 | 1 | 3 | 0 | 0 | 0 | ||
30 | 3 | 2 | 0 | 0 | 0 | ||
Lentivirus- mediated delivery system | hTERT | 6 | 2 | 1 | 1 | 0 | 0 |
9 | 1 | 2 | 1 | 1 | 0 | ||
9 | 3 | 1 | o | 2 | 0 | ||
bTERT | 1 | 3 | 2 | 4 | 1 | 1 | |
3 | 3 | 4 | 1 | 3 | 0 | ||
4 | 2 | 0 | 2 | 2 | 2 | ||
CRISPR/Cas9 system | hTERT | 2 | 4 | 3 | 1 | 2 | 1 |
3 | 2 | 1 | 2 | 1 | 2 | ||
2 | 2 | 3 | 4 | 3 | 1 | ||
bTERT | 0 | 1 | 2 | 3 | 3 | 4 | |
1 | 0 | 1 | 3 | 2 | 3 | ||
0 | 0 | 3 | 4 | 2 | 7 |
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Zhang, Z.; Han, Z.; Guo, Y.; Liu, X.; Gao, Y.; Zhang, Y. Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells. Int. J. Mol. Sci. 2021, 22, 12540. https://doi.org/10.3390/ijms222212540
Zhang Z, Han Z, Guo Y, Liu X, Gao Y, Zhang Y. Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells. International Journal of Molecular Sciences. 2021; 22(22):12540. https://doi.org/10.3390/ijms222212540
Chicago/Turabian StyleZhang, Zihan, Zhuo Han, Ying Guo, Xin Liu, Yuanpeng Gao, and Yong Zhang. 2021. "Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells" International Journal of Molecular Sciences 22, no. 22: 12540. https://doi.org/10.3390/ijms222212540