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The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development

  • Max S. FarnworthEmail author
  • Kolja N. Eckermann
  • Hassan M. M. Ahmed
  • Dominik S. Mühlen
  • Bicheng He
  • Gregor BucherEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)

Abstract

With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.

Keywords

CRISPR/Cas9 Genome engineering HDR NHEJ Tribolium Neural lineage Brain development 2A peptide 

Notes

Acknowledgements

We express our gratitude to Prof. Martin Klingler for discussions on the gene-specific enhancer trap strategy and Dr. Stefan Dippel for discussions on the bicistronic line strategy. In addition, we want to thank Patricio Ferrer Murguia for useful additional information.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Max S. Farnworth
    • 1
    • 2
    Email author
  • Kolja N. Eckermann
    • 2
    • 3
  • Hassan M. M. Ahmed
    • 3
    • 4
  • Dominik S. Mühlen
    • 1
    • 2
  • Bicheng He
    • 1
  • Gregor Bucher
    • 1
    Email author
  1. 1.Department of Evolutionary Developmental Genetics, Johann-Friedrich-Blumenbach InstituteGZMB, University of GöttingenGöttingenGermany
  2. 2.Göttingen Graduate Center for Molecular Biosciences, Neurosciences and BiophysicsGöttingenGermany
  3. 3.Department of Developmental Biology, Johann-Friedrich-Blumenbach InstituteGZMB, University of GöttingenGöttingenGermany
  4. 4.Department of Crop Protection, Faculty of AgricultureUniversity of Khartoum, Khartoum-NorthKhartoumSudan

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