Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Goat CSN1S1 Promoter Region Cloning and Bioinformatics Analysis
2.3. Vector Construction
2.4. Cell Culture
2.5. Cell Treatment
2.6. Luciferase Assays
2.7. RNA Extraction and Quantitative Real-Time PCR
2.8. Western Blot
2.9. Chromatin Immunoprecipitation (ChIP) Assays
2.10. Statistical Analysis
3. Results
3.1. Cloning and Characterization of the Goat CSN1S1 Promoter
3.2. Core Promoter Region Identification of CSN1S1 Gene
3.3. STAT5 Upregulates CSN1S1 Expression
3.4. Involvement of STAT5 in Transcriptional Regulation of CSN1S1
3.5. Prolactin Increases CSN1S1 Promoter Activity via STAT5
3.6. STAT5 Binds to the GAS Sites of CSN1S1 Promoter Region
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primers | Primer Sequence † (5′–3′) | Binding Region |
---|---|---|
Cloning and 5′ deletion primers | ||
Forward 1 | CGGGGTACCGGTGGCAAGAATAGTATTACAG | −2018 |
Forward 2 | CGGGGTACCGACATCACTTTACTGATAAG | −1715 |
Forward 3 | CGGGGTACCCAGATACACCTCTGGGAAAG | −1354 |
Forward 4 | CGGGGTACCGATTCCTTTCTTATAAACAA | −1049 |
Forward 5 | CGGGGTACCAACTTAGTGACTAAACAACA | −598 |
Forward 6 | CGGGGTACCCTTCTCATGGTCTTGTACAT | −346 |
Forward 7 | CGGGGTACCGTAAACTCTCCTCAGAATTTC | −110 |
Forward 8 | CGGGGTACCGTTAAACTGAAACCACAAAAT | −18 |
Reverse | CCCAAGCTTCAACTGCGTATTAGTGAAGA | +183 |
Site-directed mutagenesis primers ‡ | ||
GAS1-mut | CCTATGTGAGAGAAcgtTTAtgcTTTAAA | −46 |
GAS1-anti-mut | TAGGTTTATTTAAAgcaTAAacgTTCTCT | −46 |
GAS2-mut | ACTCTCCTCAGAATcgtTTGtgcGAGGAA | −92 |
GAS2-anti-mut | CTGTTCAGTTCCTCgcaCAAacgATTCTG | −92 |
GAS3-mut | AAGTCCAGATACACgcaTGGacgAGACAAAA | −1345 |
GAS3-anti-mut | TACTCGACTTTTGTCTcgtCCAtgcGTGTAT | −1345 |
GenBank ID | Gene | Primer Sequence (5′–3′) | Length (bp) |
---|---|---|---|
XM_018049127.1 | CSN1S1 | F, TCCACTAGGCACACAATACACTGA | 61 |
R, GCCAATGGGATTAGGGATGTC | |||
XM_018065112.1 | STAT5a | F, CCATCGACCTGGACAATCCC | 96 |
R, CGACTTGGTGCTCTGCCTTCTT | |||
XM_005700842.2 | UXT | F, CAGCTGGCCAAATACCTTCAA | 125 |
R, GTGTCTGGGACCACTGTGTCAA | |||
XM_005709411.1 | RPS9 | F, CCTCGACCAAGAGCTGAAG | 64 |
R, CCTCCAGACCTCACGTTTGTTC | |||
ChIP primers | GAS1 | F, GAGGAACTGAACAGAACATC | 97 |
R, GTAAAATGCTAATTTTGTGG | |||
GAS2 | F, ACAATGCCATTCCATTTCCTAT | 98 | |
R, AGGAAATCGATGTTCTGTTCAG |
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Song, N.; Luo, J.; Huang, L.; Zang, S.; He, Q.; Wu, J.; Huang, J. Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells. Foods 2022, 11, 346. https://doi.org/10.3390/foods11030346
Song N, Luo J, Huang L, Zang S, He Q, Wu J, Huang J. Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells. Foods. 2022; 11(3):346. https://doi.org/10.3390/foods11030346
Chicago/Turabian StyleSong, Ning, Jun Luo, Lian Huang, Saige Zang, Qiuya He, Jiao Wu, and Jiangtao Huang. 2022. "Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells" Foods 11, no. 3: 346. https://doi.org/10.3390/foods11030346