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Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Richard Nelson Hall, Uri Weill, Leonard Drees, Sergio Leal-Ortiz, Hongquan Li, Chew Chai, Alejandro Sánchez Alvarado, Nicholas A. Melosh, Andrew Z. Fire, Jochen C. Rink, Bo Wang
doi: https://doi.org/10.1101/2021.04.20.440701
Richard Nelson Hall
1Department of Bioengineering, Stanford University, Stanford, CA, USA
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Uri Weill
2Department of Tissue Dynamics and Regeneration, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
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Leonard Drees
2Department of Tissue Dynamics and Regeneration, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
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Sergio Leal-Ortiz
3Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA
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Hongquan Li
4Department of Electrical Engineering, Stanford University, Stanford, CA, USA
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Chew Chai
1Department of Bioengineering, Stanford University, Stanford, CA, USA
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Alejandro Sánchez Alvarado
5Stowers Institute for Medical Research, Kansas City, MO, USA
6Howard Hughes Medical Institute, Kansas City, MO, USA.
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Nicholas A. Melosh
3Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA
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Andrew Z. Fire
7Department of Pathology, Stanford, CA, USA
8Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA
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Jochen C. Rink
2Department of Tissue Dynamics and Regeneration, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
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  • For correspondence: jochen.rink@mpinat.mpg.de wangbo@stanford.edu
Bo Wang
1Department of Bioengineering, Stanford University, Stanford, CA, USA
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  • For correspondence: jochen.rink@mpinat.mpg.de wangbo@stanford.edu
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Abstract

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system.

Motivation The study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • This version of the paper reflects major changes made during the revision process at Cell Reports Methods. Significant new results include single-cell quantification of viability and transfection efficiency (Figure 2E-F), transfection and expression kinetics of in-vitro transfected cells (Figure 4), and live imaging of transfected animals (Figure 6).

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 28, 2022.
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Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection
Richard Nelson Hall, Uri Weill, Leonard Drees, Sergio Leal-Ortiz, Hongquan Li, Chew Chai, Alejandro Sánchez Alvarado, Nicholas A. Melosh, Andrew Z. Fire, Jochen C. Rink, Bo Wang
bioRxiv 2021.04.20.440701; doi: https://doi.org/10.1101/2021.04.20.440701
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Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection
Richard Nelson Hall, Uri Weill, Leonard Drees, Sergio Leal-Ortiz, Hongquan Li, Chew Chai, Alejandro Sánchez Alvarado, Nicholas A. Melosh, Andrew Z. Fire, Jochen C. Rink, Bo Wang
bioRxiv 2021.04.20.440701; doi: https://doi.org/10.1101/2021.04.20.440701

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