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Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines

SIMPLE SUMMARY: Bombyx mori is a lepidopteran insect with economic value. Its genetic background is clear, and genome sequence is relatively complete, but the function of many genes has not been determined. The genetic code expansion system has become an important means of gene function research. In...

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Autores principales: Lu, Wei, Wang, Ruolin, Wang, Pan, Ma, Sanyuan, Xia, Qingyou
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8709394/
https://www.ncbi.nlm.nih.gov/pubmed/34940169
http://dx.doi.org/10.3390/insects12121081
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author Lu, Wei
Wang, Ruolin
Wang, Pan
Ma, Sanyuan
Xia, Qingyou
author_facet Lu, Wei
Wang, Ruolin
Wang, Pan
Ma, Sanyuan
Xia, Qingyou
author_sort Lu, Wei
collection PubMed
description SIMPLE SUMMARY: Bombyx mori is a lepidopteran insect with economic value. Its genetic background is clear, and genome sequence is relatively complete, but the function of many genes has not been determined. The genetic code expansion system has become an important means of gene function research. In this study, a genetic code expansion system suitable for B. mori cells was established. This system included a modified tRNA(Pyl)/Pyrrolysyl-tRNA synthetase (PylRS) pair from Methanosarcina mazei, the reporter gene D[TAG]G formed by DsRed and EGFP through amber stop codon TAG connection and the unnatural amino acid H-Lys(Boc)-OH. In silkworm BmE and BmNs cell lines, the reporter gene expression was strictly controlled by H-Lys(Boc)-OH in the presence of both PylRS and tRNA(Pyl). The silkworm genetic code expansion system established here is another useful controllable gene expression system besides tetracycline induced expression system. ABSTRACT: Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNA(Pyl)/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNA(Pyl). Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.
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spelling pubmed-87093942021-12-25 Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines Lu, Wei Wang, Ruolin Wang, Pan Ma, Sanyuan Xia, Qingyou Insects Article SIMPLE SUMMARY: Bombyx mori is a lepidopteran insect with economic value. Its genetic background is clear, and genome sequence is relatively complete, but the function of many genes has not been determined. The genetic code expansion system has become an important means of gene function research. In this study, a genetic code expansion system suitable for B. mori cells was established. This system included a modified tRNA(Pyl)/Pyrrolysyl-tRNA synthetase (PylRS) pair from Methanosarcina mazei, the reporter gene D[TAG]G formed by DsRed and EGFP through amber stop codon TAG connection and the unnatural amino acid H-Lys(Boc)-OH. In silkworm BmE and BmNs cell lines, the reporter gene expression was strictly controlled by H-Lys(Boc)-OH in the presence of both PylRS and tRNA(Pyl). The silkworm genetic code expansion system established here is another useful controllable gene expression system besides tetracycline induced expression system. ABSTRACT: Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNA(Pyl)/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNA(Pyl). Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm. MDPI 2021-12-01 /pmc/articles/PMC8709394/ /pubmed/34940169 http://dx.doi.org/10.3390/insects12121081 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lu, Wei
Wang, Ruolin
Wang, Pan
Ma, Sanyuan
Xia, Qingyou
Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title_full Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title_fullStr Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title_full_unstemmed Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title_short Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines
title_sort genetic code expansion system for tight control of gene expression in bombyx mori cell lines
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8709394/
https://www.ncbi.nlm.nih.gov/pubmed/34940169
http://dx.doi.org/10.3390/insects12121081
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