Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)

This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate...

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Detalles Bibliográficos
Autores principales: Rincon Fernandez Pacheco, David, Sabet, Sara, Breunig, Joshua J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757563/
https://www.ncbi.nlm.nih.gov/pubmed/33377093
http://dx.doi.org/10.1016/j.xpro.2020.100199
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author Rincon Fernandez Pacheco, David
Sabet, Sara
Breunig, Joshua J.
author_facet Rincon Fernandez Pacheco, David
Sabet, Sara
Breunig, Joshua J.
author_sort Rincon Fernandez Pacheco, David
collection PubMed
description This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort(m4(ACTB-tdTomato, EGFP)Luo/)J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).
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spelling pubmed-77575632020-12-28 Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR) Rincon Fernandez Pacheco, David Sabet, Sara Breunig, Joshua J. STAR Protoc Protocol This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort(m4(ACTB-tdTomato, EGFP)Luo/)J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019). Elsevier 2020-12-09 /pmc/articles/PMC7757563/ /pubmed/33377093 http://dx.doi.org/10.1016/j.xpro.2020.100199 Text en © 2020 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Rincon Fernandez Pacheco, David
Sabet, Sara
Breunig, Joshua J.
Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title_full Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title_fullStr Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title_full_unstemmed Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title_short Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR)
title_sort preparation, assembly, and transduction of transgenic elements using mosaic analysis with dual recombinases (madr)
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757563/
https://www.ncbi.nlm.nih.gov/pubmed/33377093
http://dx.doi.org/10.1016/j.xpro.2020.100199
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