Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
Abstract
:1. Introduction
2. Results
2.1. Plasmid Construction and mRNA Synthesis
2.2. Optimization of Transfection Conditions
2.3. Generation of mRNA Induced Pluripotent Stem Cells (mRNA iPS)
2.4. Characterization and Identification of the Generated mRNA iPSCs
2.5. Genetic and Epigenetic Changes of the Reprogramming Factors and Pluripotency Markers during mRNA iPS Generation
3. Discussion
4. Materials and Methods
4.1. RNA Extraction and cDNA Amplification
4.2. Plasmid Construction
Gene | Accession No. | ORF Primers |
---|---|---|
Oct4 | NM_013633 | F: 5'CGGAATTCCGCCACCTTCCCCATGGCTGGACACC3'
R: 5'CCCTCGAGGGTGATCAACAGCATCACTGAGCTTC3' |
Sox2 | NM_011443 | F: 5'CGGAATTCCGATGTATAACATGATGGAGACGGAGCT3'
R: 5'CCCTCGAGGGTCACATGTGCGACAGGGGCAGT3' |
c-Myc | NM_001177352 | F: 5'CGGAATTCCGATGCCCCTCAACGTGAACTTCACC3'
R: 5'CCCTCGAGGGTTATGCACCAGAGTTTCGAAGC3' |
Klf4 | NM_010637 | F: 5'CGGAATTCCGATGAGGCAGCCACCTGGCGAGT3' R: 5'CCCTCGAGGGCTACGTGGGATTTAAAAGTGCCTC3' |
4.3. In Vitro Transcription of mRNA
4.4. Mouse Embryonic Fibroblast (MEF) Isolation
4.5. Cell Culture
4.6. Cell Transfection
4.7. Quantitative Real-Time PCR (qPCR)
Gene | Forward Primer | Reverse Primer | References | |
---|---|---|---|---|
Oct4 | Total | 5'CAGACCACCATCTGTCGCTTC3' | 5'AGACTCCACCTCACACGGTTCTC3' | This study |
Endogenous | 5'TCTTTCCACCAGGCCCCCGGCTC3' | 5'TGCGGGCGGACATGGGGAGATCC3' | [11] | |
Sox2 | Total | 5'GGTTACCTCTTCCTCCCACTCCAG3' | 5'TCACATGTGCGACAGGGGCAG3' | |
Endogenous | 5'TAGAGCTAGACTCCGGGCGATGA3' | 5'TTGCCTTAAACAAGACCACGAAA3' | ||
c-Myc | Total | 5'CCTAGTGCTGCATGAGGAGACAC3' | 5'TCCACAGACACCACATCAATTTCTT3' | This study |
Endogenous | 5'TGACCTAACTCGAGGAGGAGCTGGAATC3' | 5'AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC3' | [11] | |
Klf4 | Total | 5'ACAGCCACCCACACTTGTGACTA3' | 5'GGCGAATTTCCACCCACAG3' | This study |
Endogenous | 5'GCGAACTCACACAGGCGAGAAACC3' | 5'TCGCTTCCTCTTCCTCCGACACA3' | [11] | |
Gapdh | – | 5'TGTGTCCGTCGTGGATCTGA3' | 5'TTGCTGTTGAAGTCGCAGGAG3' | This study |
4.8. Immunofluorescence
4.9. Fluorescence Activated Cell Sorting (FACS)
4.10. Alkaline Phosphatase Staining
4.11. Bisulfite Genomic Sequencing
Gene | Forward Primer | Reverse Primer |
---|---|---|
Nanog | 5'GATTTTGTAGGTGGGATTAATTGTGAATTT3' | 5'ACCAAAAAAACCCACACTCATATCAATATA3' |
Oct4 | F1: 5'GTTGTTTTGTTTTGGTTTTGGATAT3' F2: 5'ATGGGTTGAAATATTGGGTTTATTTA3' | 5'CCACCCTCTAACCTTAACCTCTAAC3' |
4.12. In Vitro Differentiation of mRNA iPSCs
Gene | Accession No. | Primers |
---|---|---|
Sox1 | NM_009233 | F: 5'GGATCTCTGGTCAAGTCGGAG3'
R: 5'CTGGCGCTCGGCTCTCCAGAG3' |
Nestin | NM_016701 | F: 5' TCTGGAAGTCAACAGAGGTGG3'
R: 5'ACGGAGTCTTGTTCACCTGC3' |
α-SMA | NM_007392 | F: 5'GAGAAGAGCTACGAACTGCCTGAC3'
R: 5'CACATCTGCTGGAAGGTAGACAG3' |
Bra | NM_009309 | F: 5'GTTCCTGGTGCTGGCACCCTCTGC3'
R: 5'CAGACCAGAGACTGGGATACTG3' |
Sox17 | NM_011441 | F: 5'CACAGCAGAACCCAGATCTGCA3'
R: 5'CATGTGCGGAGACATCAGCGGAG3' |
AFP | NM_007423 | F: 5'GTGAGCATTGCCTCCACGTGCTG3'
R: 5'GTGACAGCCGCCAGCTGCTCCTC3' |
4.13. Teratoma Formation and Histological Analysis
4.14. Karyotyping Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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El-Sayed, A.K.; Zhang, Z.; Zhang, L.; Liu, Z.; Abbott, L.C.; Zhang, Y.; Li, B. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors. Int. J. Mol. Sci. 2014, 15, 21840-21864. https://doi.org/10.3390/ijms151221840
El-Sayed AK, Zhang Z, Zhang L, Liu Z, Abbott LC, Zhang Y, Li B. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors. International Journal of Molecular Sciences. 2014; 15(12):21840-21864. https://doi.org/10.3390/ijms151221840
Chicago/Turabian StyleEl-Sayed, Ahmed Kamel, Zhentao Zhang, Lei Zhang, Zhiyong Liu, Louise C. Abbott, Yani Zhang, and Bichun Li. 2014. "Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors" International Journal of Molecular Sciences 15, no. 12: 21840-21864. https://doi.org/10.3390/ijms151221840