Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Book cover Brain Development

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Perry M, Konstantinides N, Pinto-Teixeira F, Desplan C (2017) Generation and evolution of neural cell types and circuits: insights from the Drosophila visual system. Annu Rev Genet 51:501–527

    Article  CAS  Google Scholar 

  2. Doe CQ (2017) Temporal patterning in the Drosophila CNS. Annu Rev Cell Dev Biol 33:219–240

    Article  CAS  Google Scholar 

  3. Urbach R, Technau GM (2004) Neuroblast formation and patterning during early brain development in Drosophila. BioEssays 26:739–751

    Article  CAS  Google Scholar 

  4. Hartenstein V, Stollewerk A (2015) The evolution of early neurogenesis. Dev Cell 32:390–407

    Article  CAS  Google Scholar 

  5. Arendt D, Tosches MA, Marlow H (2016) From nerve net to nerve ring, nerve cord and brain—evolution of the nervous system. Nat Rev Neurosci 17:61–72

    Article  CAS  Google Scholar 

  6. El Jundi B, Heinze S Three-dimensional atlases of insect brains. Neurohistology and Imaging: Basic Techniques

    Google Scholar 

  7. Koniszewski NDB, Kollmann M, Bigham M, Farnworth M, He B, Büscher M, Hütteroth W, Binzer M, Schachtner J, Bucher G (2016) The insect central complex as model for heterochronic brain development—background, concepts, and tools. Dev Genes Evol 226:209–219

    Article  CAS  Google Scholar 

  8. Brown SJ, Mahaffey JP, Lorenzen MD, Denell RE, Mahaffey JW (1999) Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev 1:11–15

    Article  CAS  Google Scholar 

  9. Bucher G, Scholten J, Klingler M (2002) Parental RNAi in Tribolium (Coleoptera). Curr Biol 12:R85–R86

    Article  CAS  Google Scholar 

  10. Tomoyasu Y, Denell RE (2004) Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Dev Genes Evol 214:575–578

    Article  CAS  Google Scholar 

  11. Schmitt-Engel C, Schultheis D, Schwirz J et al (2015) The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun 6:7822

    Article  CAS  Google Scholar 

  12. Berghammer AJ, Klingler M, Wimmer EA (1999) Genetic techniques: A universal marker for transgenic insects. Nature 402:370–371

    Article  CAS  Google Scholar 

  13. Trauner J, Schinko J, Lorenzen MD, Shippy TD, Wimmer EA, Beeman RW, Klingler M, Bucher G, Brown SJ (2009) Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps. BMC Biol 7:73

    Article  Google Scholar 

  14. Lorenzen MD, Kimzey T, Shippy TD, Brown SJ, Denell RE, Beeman RW (2007) piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Mol Biol 16:265–275

    Article  CAS  Google Scholar 

  15. Gilles AF, Schinko JB, Averof M (2015) Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum. Development 142:2832–2839

    Article  CAS  Google Scholar 

  16. Gilles AF, Schinko JB, Schacht MI, Enjolras C, Averof M (2019) Clonal analysis by tunable CRISPR-mediated excision. Development 146 (1):dev170969

    Article  Google Scholar 

  17. Hayashi S, Ito K, Sado Y et al (2002) GETDB, a database compiling expression patterns and molecular locations of a collection of gal4 enhancer traps. Genesis 34:58–61

    Article  CAS  Google Scholar 

  18. Mollereau B, Wernet MF, Beaufils P, Killian D, Pichaud F, Kühnlein R, Desplan C (2000) A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells. Mech Dev 93:151–160

    Article  CAS  Google Scholar 

  19. O’Kane CJ, Gehring WJ (1987) Detection in situ of genomic regulatory elements in Drosophila. PNAS 84:9123–9127

    Article  Google Scholar 

  20. Pfeiffer BD, Jenett A, Hammonds AS et al (2008) Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci U S A 105:9715–9720

    Article  CAS  Google Scholar 

  21. Wu JS, Luo L (2006) A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat Protoc 1:2110–2115

    Article  CAS  Google Scholar 

  22. Jin EJ, Kiral FR, Ozel MN, Burchardt LS, Osterland M, Epstein D, Wolfenberg H, Prohaska S, Hiesinger PR (2018) Live Observation of Two Parallel Membrane Degradation Pathways at Axon Terminals. Curr Biol 28:1027–1038.e4

    Article  CAS  Google Scholar 

  23. Eckert C, Aranda M, Wolff C, Tautz D (2004) Separable stripe enhancer elements for the pair-rule gene hairy in the beetle Tribolium. EMBO Rep 5:638–642

    Article  CAS  Google Scholar 

  24. Wilson C, Bellen H, Gehring W (1990) Position Effects on Eukaryotic Gene-Expression. Annu Rev Cell Biol 6:679–714

    Article  CAS  Google Scholar 

  25. John AV, Sramkoski LL, Walker EA, Cooley AM, Wittkopp PJ (2016) Sensitivity of allelic divergence to genomic position: Lessons from the Drosophila tan gene. G3 6:2955–2962

    Article  Google Scholar 

  26. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A Programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  Google Scholar 

  27. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278

    Article  CAS  Google Scholar 

  28. Gratz SJ, Wildonger J, Harrison MM, O’Connor-Giles KM (2013) CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly 7:249–255

    Article  CAS  Google Scholar 

  29. Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096

    Article  Google Scholar 

  30. Rylee JC, Siniard DJ, Doucette K, Zentner GE, Zelhof AC (2018) Expanding the genetic toolkit of Tribolium castaneum. PLoS One 13:e0195977

    Article  Google Scholar 

  31. Dönitz J, Schmitt-Engel C, Grossmann D, Gerischer L, Tech M, Schoppmeier M, Klingler M, Bucher G (2015) iBeetle-Base: a database for RNAi phenotypes in the red flour beetle Tribolium castaneum. Nucl Acids Res 43:D720–D725

    Article  Google Scholar 

  32. Dönitz J, Gerischer L, Hahnke S, Pfeiffer S, Bucher G (2018) Expanded and updated data and a query pipeline for iBeetle-Base. Nucleic Acids Res 46:D831–D835

    Article  Google Scholar 

  33. Lai Y-T, Deem KD, Borràs-Castells F, Sambrani N, Rudolf H, Suryamohan K, El-Sherif E, Halfon MS, McKay DJ, Tomoyasu Y (2018) Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum. Development. https://doi.org/10.1242/dev.160663

    Article  Google Scholar 

  34. Häcker U, Nystedt S, Barmchi MP, Horn C, Wimmer EA (2003) piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila. PNAS 100:7720–7725

    Article  Google Scholar 

  35. Kvon EZ, Kazmar T, Stampfel G, Yáñez-Cuna JO, Pagani M, Schernhuber K, Dickson BJ, Stark A (2014) Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature 512:91–95

    Article  CAS  Google Scholar 

  36. Schinko JB, Weber M, Viktorinova I, Kiupakis A, Averof M, Klingler M, Wimmer EA, Bucher G (2010) Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters. BMC Dev Biol 10:53

    Article  Google Scholar 

  37. Schinko JB, Hillebrand K, Bucher G (2012) Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum. Dev Genes Evol 222:287–298

    Article  CAS  Google Scholar 

  38. Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479

    Article  CAS  Google Scholar 

  39. Lorenzen MD, Brown SJ, Denell RE, Beeman RW (2002) Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase. Genetics 160:225–234

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Sarov M, Barz C, Jambor H et al (2016) A genome-wide resource for the analysis of protein localisation in Drosophila. elife 5:e12068

    Article  Google Scholar 

  41. Donnelly MLL, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82:1013–1025

    Article  CAS  Google Scholar 

  42. Szymczak-Workman AL, Vignali KM, Vignali DAA (2012) Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012. pdb.ip067876-pdb.ip067876

    Google Scholar 

  43. Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, Kim M-K, Shin BA, Choi S-Y (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6:e18556

    Article  CAS  Google Scholar 

  44. Brown SJ, Shippy TD, Miller S, Bolognesi R, Beeman RW, Lorenzen MD, Bucher G, Wimmer EA, Klingler M (2009) The red flour beetle, Tribolium castaneum (Coleoptera): a model for studies of development and pest biology. Cold Spring Harb Protoc 2009:pdb.emo126

    Article  Google Scholar 

  45. Tribolium Genome Sequencing Consortium, Richards S, Gibbs RA, et al (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452:949–955

    Google Scholar 

  46. Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196:961–971

    Article  CAS  Google Scholar 

  47. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

    Article  CAS  Google Scholar 

  48. Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832

    Article  CAS  Google Scholar 

  49. Horn C, Schmid BGM, Pogoda FS, Wimmer EA (2002) Fluorescent transformation markers for insect transgenesis. Insect Biochem Mol Biol 32:1221–1235

    Article  CAS  Google Scholar 

  50. Ulrich A, Andersen KR, Schwartz TU (2012) Exponential megapriming PCR (EMP) cloning—seamless DNA insertion into any target plasmid without sequence constraints. PLoS One 7:e53360

    Article  CAS  Google Scholar 

  51. Beumer KJ, Trautman JK, Mukherjee K, Carroll D (2013) Donor DNA utilization during gene targeting with zinc-finger nucleases. G3 3:657–664

    Google Scholar 

  52. Tycko J, Myer VE, Hsu PD (2016) Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell 63:355–370

    Article  CAS  Google Scholar 

  53. Berghammer A, Bucher G, Maderspacher F, Klingler M (1999) A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum. Dev Genes Evol 209:382–389

    Article  CAS  Google Scholar 

  54. Posnien N, Schinko J, Grossmann D, Shippy TD, Konopova B, Bucher G (2009) RNAi in the red flour beetle (Tribolium). Cold Spring Harb Protoc 2009:pdb.prot5256

    Article  Google Scholar 

  55. Eckermann KN, Ahmed HMM, KaramiNejadRanjbar M, Dippel S, Ogaugwu CE, Kitzmann P, Isah MD, Wimmer EA (2018) Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species. Insect Biochem Mol Biol 98:16–24

    Article  CAS  Google Scholar 

  56. Strobl F, Ross JA, Stelzer EHK (2017) Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue. PLoS One 12:e0182564

    Article  Google Scholar 

  57. Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 163:759–771

    Article  CAS  Google Scholar 

  58. Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44:W272–W276

    Article  CAS  Google Scholar 

  59. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407

    Article  CAS  Google Scholar 

  60. Clarke R, Heler R, MacDougall MS, Yeo NC, Chavez A, Regan M, Hanakahi L, Church GM, Marraffini LA, Merrill BJ (2018) Enhanced bacterial immunity and mammalian genome editing via rna-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Mol Cell 71:42–55. e8

    Article  CAS  Google Scholar 

  61. Port F, Chen H-M, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci 111:E2967–E2976

    Article  CAS  Google Scholar 

  62. Bassett AR, Tibbit C, Ponting CP, Liu J-L (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4:220–228

    Article  CAS  Google Scholar 

  63. Keeler KJ, Dray T, Penney JE, Gloor GB (1996) Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster. Mol Cell Biol 16:522–528

    Article  CAS  Google Scholar 

  64. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen Z-Y, Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Evolutionary Developmental Genetics, Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany

    Max S. Farnworth, Dominik S. Mühlen, Bicheng He & Gregor Bucher

  2. Göttingen Graduate Center for Molecular Biosciences, Neurosciences and Biophysics, Göttingen, Germany

    Max S. Farnworth, Kolja N. Eckermann & Dominik S. Mühlen

  3. Department of Developmental Biology, Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany

    Kolja N. Eckermann & Hassan M. M. Ahmed

  4. Department of Crop Protection, Faculty of Agriculture, University of Khartoum, Khartoum-North, Khartoum, Sudan

    Hassan M. M. Ahmed

Authors
  1. Max S. Farnworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kolja N. Eckermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hassan M. M. Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dominik S. Mühlen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bicheng He
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gregor Bucher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Max S. Farnworth or Gregor Bucher .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G. Sprecher

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Farnworth, M.S., Eckermann, K.N., Ahmed, H.M.M., Mühlen, D.S., He, B., Bucher, G. (2020). The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation