Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line
Abstract
:1. Introduction
2. Results
2.1. Effect of First Intron on Chicken Growth Hormone (cGH) Complete Coding Sequences (CDSs) Expression In Vitro
2.2. Effect of Intron 1 Fragments on Reporter Gene Expression In Vitro
2.3. Transcriptional Factor Binding Sites (TFBSs) in Intron 1 of the cGH Gene
2.4. Role of the Intronic Negative Glucocorticoid Response Element (nGRE) in Reporter Gene Expression In Vitro
2.5. Effect of the Length of GH Intron 1 Fragments on Reporter Gene Expression In Vitro
3. Discussion
4. Materials and Methods
4.1. Construction of Plasmids
4.2. Cell Cultures
4.3. Cell Transfection
4.4. RNA Isolation, RT-PCR and Real-Time Quantitative PCR (RT-qPCR)
4.5. Dual-Luciferase Reporter Assay
4.6. Immunoblotting
4.7. Electrophoretic Mobility Shift Assay (EMSA)
4.8. Statistical Analysis
4.9. Ethics Standards
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Position | Name |
---|---|
+55/+372 | AP1FJ, AP1, AP4, AML1, CAP, CDXA, CEBPB, CEBP, CREL, GATA3, GATA2, GATA1, GR, LMO2COM, MZF1, OCT1, P53, SRY, SP1, TST1 |
+372/+485 | CAP, CDXA, CETS1P54, DELTAEF1, MYOD, NKX25, SRY, TATA |
+485/+684 | AP4, AML1, CAP, CETS1P54, CEBPB, DELTAEF1, E47, GRE, LMO2COM, MZF1, NKX25, SRY, USF |
+684/+969 | AP4, CAP, CEBPB, E2F, E47, HNF3β, IK2, LMO2COM, MZF1, MYB, STAT, USF |
Groups | Length (bp) | Mean mRNA Expression Level ± SEM | Mean Protein Expression Level ± SEM |
---|---|---|---|
pGL3-cGH-in4 | 915 | 1.159 ± 0.0201 | 0.252 ± 0.0313 |
pGL3-cGH-in7 | 897 | 1.716 ± 0.1975 | 1.223 ± 0.3150 |
pGL3-cGH-in6 | 715 | 1.638 ± 0.1396 | 1.054 ± 0.1044 |
pGL3-cGH-in3 | 598 | 2.992 ± 0.0904 | 1.79 ± 0.4998 |
pGL3-cGH-in2 | 485 | 0.878 ± 0.0238 | 0.429 ± 0.0386 |
pGL3-cGH-in1 | 286 | 9.681 ± 0.0885 | 1.344 ± 0.2907 |
pGL3-cGH-in5 | 200 | 1.649 ± 0.1000 | 0.632 ± 0.0640 |
pGL3-control | 0 | 1.007 ± 0.0224 | 1.003 ± 0.0436 |
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Ma, J.-E.; Lang, Q.-Q.; Qiu, F.-F.; Zhang, L.; Li, X.-G.; Luo, W.; Wang, J.; Wang, X.; Lin, X.-R.; Liu, W.-S.; et al. Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line. Int. J. Mol. Sci. 2016, 17, 1863. https://doi.org/10.3390/ijms17111863
Ma J-E, Lang Q-Q, Qiu F-F, Zhang L, Li X-G, Luo W, Wang J, Wang X, Lin X-R, Liu W-S, et al. Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line. International Journal of Molecular Sciences. 2016; 17(11):1863. https://doi.org/10.3390/ijms17111863
Chicago/Turabian StyleMa, Jing-E., Qian-Qian Lang, Feng-Fang Qiu, Li Zhang, Xiang-Guang Li, Wen Luo, Juan Wang, Xing Wang, Xi-Ran Lin, Wen-Sheng Liu, and et al. 2016. "Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line" International Journal of Molecular Sciences 17, no. 11: 1863. https://doi.org/10.3390/ijms17111863