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Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines

We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, and partial recombination using both approaches. Reviewing the literature gave us the impression tha...

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Autores principales: Tromp, Alisha, Wang, Haitao, Hall, Thomas E., Mowry, Bryan, Giacomotto, Jean
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10433388/
https://www.ncbi.nlm.nih.gov/pubmed/37601640
http://dx.doi.org/10.3389/fphys.2023.1221310
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author Tromp, Alisha
Wang, Haitao
Hall, Thomas E.
Mowry, Bryan
Giacomotto, Jean
author_facet Tromp, Alisha
Wang, Haitao
Hall, Thomas E.
Mowry, Bryan
Giacomotto, Jean
author_sort Tromp, Alisha
collection PubMed
description We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, and partial recombination using both approaches. Reviewing the literature gave us the impression that these issues are common among the zebrafish community using the Cre/Lox system. While some researchers took advantage of these problems for specific applications, such as cell and lineage tracing using the Zebrabow construct, we tried here to improve the efficiency and reliability of this system by constituting and testing a new set of tools for zebrafish genetics. First, we implemented a codon-improved Cre version (iCre) designed for rodent studies to counteract some of the aforementioned problems. This eukaryotic-like iCre version was engineered to i) reduce silencing, ii) increase mRNA stability, iii) enhance translational efficiency, and iv) improve nuclear translocation. Second, we established a new set of tol2-kit compatible vectors to facilitate the generation of either iCre-mRNA or iCre-transgenes for transient and transgenic experiments, respectively. We then validated the use of this material and are providing tips for users. Interestingly, during the validation steps, we found that maternal iCRE-mRNA and/or protein deposition from female transgenics systematically led to complete/homogeneous conversion of all tested Lox-responder-transgenes, as opposed to some residual imperfect conversion when using males-drivers or mRNA injections. Considering that we did not find any evidence of Cre-protein soaking and injections in the literature as it is usually conducted with cells, we tested these approaches. While soaking of cell-permeant CRE-protein did not lead to any detectable Lox-conversion, 1ng–10 ng protein injections led to robust and homogeneous Lox-recombination, suggesting that the use of protein could be a robust option for exogenous delivery. This approach may be particularly useful to manipulate housekeeping genes involved in development, sex determination and reproduction which are difficult to investigate with traditional knockout approaches. All in all, we are providing here a new set of tools that should be useful in the field.
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spelling pubmed-104333882023-08-18 Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines Tromp, Alisha Wang, Haitao Hall, Thomas E. Mowry, Bryan Giacomotto, Jean Front Physiol Physiology We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, and partial recombination using both approaches. Reviewing the literature gave us the impression that these issues are common among the zebrafish community using the Cre/Lox system. While some researchers took advantage of these problems for specific applications, such as cell and lineage tracing using the Zebrabow construct, we tried here to improve the efficiency and reliability of this system by constituting and testing a new set of tools for zebrafish genetics. First, we implemented a codon-improved Cre version (iCre) designed for rodent studies to counteract some of the aforementioned problems. This eukaryotic-like iCre version was engineered to i) reduce silencing, ii) increase mRNA stability, iii) enhance translational efficiency, and iv) improve nuclear translocation. Second, we established a new set of tol2-kit compatible vectors to facilitate the generation of either iCre-mRNA or iCre-transgenes for transient and transgenic experiments, respectively. We then validated the use of this material and are providing tips for users. Interestingly, during the validation steps, we found that maternal iCRE-mRNA and/or protein deposition from female transgenics systematically led to complete/homogeneous conversion of all tested Lox-responder-transgenes, as opposed to some residual imperfect conversion when using males-drivers or mRNA injections. Considering that we did not find any evidence of Cre-protein soaking and injections in the literature as it is usually conducted with cells, we tested these approaches. While soaking of cell-permeant CRE-protein did not lead to any detectable Lox-conversion, 1ng–10 ng protein injections led to robust and homogeneous Lox-recombination, suggesting that the use of protein could be a robust option for exogenous delivery. This approach may be particularly useful to manipulate housekeeping genes involved in development, sex determination and reproduction which are difficult to investigate with traditional knockout approaches. All in all, we are providing here a new set of tools that should be useful in the field. Frontiers Media S.A. 2023-08-02 /pmc/articles/PMC10433388/ /pubmed/37601640 http://dx.doi.org/10.3389/fphys.2023.1221310 Text en Copyright © 2023 Tromp, Wang, Hall, Mowry and Giacomotto. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Tromp, Alisha
Wang, Haitao
Hall, Thomas E.
Mowry, Bryan
Giacomotto, Jean
Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title_full Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title_fullStr Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title_full_unstemmed Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title_short Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines
title_sort optimising the zebrafish cre/lox toolbox. codon improved icre, new gateway tools, cre protein and guidelines
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10433388/
https://www.ncbi.nlm.nih.gov/pubmed/37601640
http://dx.doi.org/10.3389/fphys.2023.1221310
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