Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A
Abstract
:1. Introduction
2. Results
2.1. Construction of Nonviral Targeting Vector minipHrn-2bF8
2.2. Gene Targeting of F8 into the rDNA Locus of HA-iPSCs Using TALENickases
2.3. Verification of F8 Expression in F8-Modified hiPSCs
2.4. Generation and Characterization of Induced HPCs (iHPCs) and Induced MKs (iMKs)
2.5. Verification of F8 Expression in F8-Modified iHPCs
2.6. Generation and Characterization of Induced MSCs (iMSCs)
2.7. Verification of FVIII Expression in F8-Modified iMSCs
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Plasmids and Gene Targeting
4.3. RT-PCR and qRT-PCR
4.4. Southern Blotting
4.5. Karyotyping
4.6. Immunofluorescence Staining
4.7. Differentiation of hiPSCs into iHPCs and iMKs
4.8. Characterization of iHPCs and iMKs
4.9. Differentiation of hiPSCs into iMSCs
4.10. Characterization and Identification of Differentiation Potential of iMSCs
4.11. FVIII Assay
4.12. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Zhao, J.; Zhou, M.; Wang, Z.; Wu, L.; Hu, Z.; Liang, D. Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A. Int. J. Mol. Sci. 2022, 23, 623. https://doi.org/10.3390/ijms23020623
Zhao J, Zhou M, Wang Z, Wu L, Hu Z, Liang D. Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A. International Journal of Molecular Sciences. 2022; 23(2):623. https://doi.org/10.3390/ijms23020623
Chicago/Turabian StyleZhao, Junya, Miaojin Zhou, Zujia Wang, Lingqian Wu, Zhiqing Hu, and Desheng Liang. 2022. "Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A" International Journal of Molecular Sciences 23, no. 2: 623. https://doi.org/10.3390/ijms23020623