Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development
| Autor: | Spenser S. Smith, Richard A. Schneider, An Nguyen, Daniel Chu, Zuzana Vavrusova |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Embryo
Nonmammalian Plasmid PiggyBac transposon Genes Reporter Gene expression Gene Order Developmental Biology (General) Cloning Molecular Promoter Regions Genetic Cells Cultured Cultured Nonmammalian Electroporation Gene Expression Regulation Developmental Embryo Cell biology PiggyBac Transposon System Avian embryos General Agricultural and Biological Sciences Biotechnology Plasmids QH301-705.5 Cells 1.1 Normal biological development and functioning Transgene Science Genetic Vectors Green Fluorescent Proteins Embryonic Development Bioengineering Biology In ovo General Biochemistry Genetics and Molecular Biology Promoter Regions Genetic Underpinning research Genetics Animals Reporter Gene Methods & Techniques Tet-inducible Molecular Gene Expression Regulation Genes DNA Transposable Elements Generic health relevance Other Biological Sciences Cloning |
| Zdroj: | Biology Open article-version (VoR) Version of Record Biology Open, Vol 9, Iss 10 (2020) Biology open, vol 9, iss 10 |
| ISSN: | 2046-6390 |
| Popis: | Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo. Summary: We have designed an optimized and integrating inducible-promoter system to control the timing, spatial domains, and levels of gene misexpression throughout avian development. |
| Databáze: | OpenAIRE |
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