Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus
Abstract
:1. Introduction
2. Results
2.1. LIN28 Knockdown in Trophectoderm Resulted in Reduced Proliferation of Trophoblast Cells and Lower Expression of Proliferation-Associated Genes
2.2. Ovine Trophoblast Cells Generated from Day 16 Trophectoderm Had a Significant Reduction in LIN28
2.3. Overexpression of LIN28 in iOTR Cells Resulted in Increased Expression of Proliferation-Associated Genes
2.4. Overexpression of LIN28 Led to Significant Increase in Trophoblast Cell Proliferation
3. Discussion
4. Materials and Methods
4.1. Lentivirus Vector Construction for shRNA Expression
4.2. Lentivirus Vector Construction for Overexpression of LIN28A and LIN28B
4.3. Lentiviral Vector for Immortalizing Passaged Ovine Trophoblast Cells
4.4. Production of Lentiviral Particles
4.5. Blastocyst Collection and Transfer
4.6. Tissue Collection
4.7. Cell Lines
4.8. Overexpression of LIN28A and LIN28B
4.9. RNA Extraction and Real-Time PCR
4.10. Protein Extraction and Western Blot
4.11. Cell Proliferation Assay
4.12. Matrigel Invasion Assay
4.13. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
OTR | non-immortalized ovine trophoblast cells |
iOTR | immortalized ovine trophoblast cells |
TE | trophectoderm |
SC | scramble control shRNA |
AKD | knockdown of LIN28A |
BKD | knockdown of LIN28B |
EVC | iOTR cells infected with empty lentiviral expression vectors |
AKI | iOTR cells overexpressing LIN28A |
BKI | iOTR cells overexpressing LIN28B |
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Ali, A.; Stenglein, M.D.; Spencer, T.E.; Bouma, G.J.; Anthony, R.V.; Winger, Q.A. Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus. Int. J. Mol. Sci. 2020, 21, 2549. https://doi.org/10.3390/ijms21072549
Ali A, Stenglein MD, Spencer TE, Bouma GJ, Anthony RV, Winger QA. Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus. International Journal of Molecular Sciences. 2020; 21(7):2549. https://doi.org/10.3390/ijms21072549
Chicago/Turabian StyleAli, Asghar, Mark D. Stenglein, Thomas E. Spencer, Gerrit J. Bouma, Russell V. Anthony, and Quinton A. Winger. 2020. "Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus" International Journal of Molecular Sciences 21, no. 7: 2549. https://doi.org/10.3390/ijms21072549