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Targeted and Random Transposon-Assisted Single-Copy Transgene Insertion in C. elegans

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C. elegans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2468))

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Abstract

Transgenesis in model organisms is an essential tool for determining the function of protein-coding genes and non-coding regulatory regions. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extra-chromosomal arrays, but transgenes in arrays are frequently mosaic, over-expressed in some tissues, and silenced in the germline. Here, we describe methods to insert single-copy transgenes into specific genomic locations (MosSCI) or random locations (miniMos) using Mos1 transposons. Single-copy insertions allow expression at endogenous levels, expression in the germline, and identification of active and repressed regions of the genome.

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References

  1. Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10:3959–3970

    Article  CAS  Google Scholar 

  2. Husson SJ, Gottschalk A, Leifer AM (2013) Optogenetic manipulation of neural activity in C. elegans: from synapse to circuits and behaviour. Biol Cell 105:235–250. https://doi.org/10.1111/boc.201200069

    Article  CAS  PubMed  Google Scholar 

  3. Kelly WG, Xu S, Montgomery MK, Fire A (1997) Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146:227–238

    Article  CAS  Google Scholar 

  4. Grishok A, Sinskey JL, Sharp PA (2005) Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev 19:683–696. https://doi.org/10.1101/gad.1247705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123–132. https://doi.org/10.1016/S0092-8674(00)81644-X

    Article  CAS  PubMed  Google Scholar 

  6. Vastenhouw NL, Fischer SEJ, Robert VJP, Thijssen KL, Fraser AG, Kamath RS, Ahringer J, Plasterk RHA (2003) A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans. Curr Biol 13:1311–1316. https://doi.org/10.1016/S0960-9822(03)00539-6

    Article  CAS  PubMed  Google Scholar 

  7. Praitis V, Casey E, Collar D, Austin J (2001) Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157:1217–1226

    Article  CAS  Google Scholar 

  8. Kage-Nakadai E, Kobuna H, Funatsu O, Otori M, Gengyo-Ando K, Yoshina S, Hori S, Mitani S (2012) Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method. BMC Biotechnol 12:1. https://doi.org/10.1186/1472-6750-12-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Paix A, Folkmann A, Rasoloson D, Seydoux G (2015) High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics 201:47–54. https://doi.org/10.1534/genetics.115.179382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods 10:1028–1034. https://doi.org/10.1038/nmeth.2641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B (2015) Streamlined genome engineering with a self-excising drug selection cassette. Genetics 200:1035–1049. https://doi.org/10.1534/genetics.115.178335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schwartz ML, Jorgensen EM (2016) SapTrap, a toolkit for high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans. Genetics 202:1277–1288. https://doi.org/10.1534/genetics.115.184275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Frøkjær-Jensen C, Wayne Davis M, Hopkins CE, Newman BJ, Thummel JM, Olesen S-P, Grunnet M, Jorgensen EM (2008) Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 40:1375–1383. https://doi.org/10.1038/ng.248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Frøkjær-Jensen C, Davis MW, Sarov M, Taylor J, Flibotte S, LaBella M, Pozniakovsky A, Moerman DG, Jorgensen EM (2014) Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods 11:529–534. https://doi.org/10.1038/nmeth.2889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bessereau J-L, Wright A, Williams DC, Schuske K, Davis MW, Jorgensen EM (2001) Mobilization of a drosophila transposon in the Caenorhabditis elegans germ line. Nature 413:70–74. https://doi.org/10.1038/35092567

    Article  CAS  PubMed  Google Scholar 

  16. Robert V, Bessereau J-L (2007) Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks. EMBO J 26:170–183. https://doi.org/10.1038/sj.emboj.7601463

    Article  CAS  PubMed  Google Scholar 

  17. Vallin E, Gallagher J, Granger L, Martin E, Belougne J, Maurizio J, Duverger Y, Scaglione S, Borrel C, Cortier E, Abouzid K, Carre-Pierrat M, Gieseler K, Ségalat L, Kuwabara PE, Ewbank JJ (2012) A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community. PLoS One 7:e30482. https://doi.org/10.1371/journal.pone.0030482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Frøkjær-Jensen C, Davis MW, Ailion M, Jorgensen EM (2012) Improved Mos1-mediated transgenesis in C. elegans. Nat Methods 9:117–118. https://doi.org/10.1038/nmeth.1865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. El Mouridi S, Peng Y, Frøkjær-Jensen C (2020) Characterizing a strong pan-muscular promoter (Pmlc-1) as a fluorescent co-injection marker to select for single-copy insertions. MicroPubl Biol 2020. https://doi.org/10.17912/micropub.biology.000302

  20. Fan X, De Henau S, Feinstein J, Miller SI, Han B, Frøkjær-Jensen C, Griffin EE (2020) SapTrap assembly of Caenorhabditis elegans MosSCI transgene vectors. G3 (Bethesda) 10:635–644. https://doi.org/10.1534/g3.119.400822

    Article  CAS  Google Scholar 

  21. Aram R, MacGillivray K, Li C, Saltzman A (2019) Tools for Mos1-mediated single copy insertion (mosSCI) with excisable unc-119(+) or NeoR (G418) selection cassettes. MicroPubl Biol 2019. https://doi.org/10.17912/micropub.biology.000146

  22. Boulin T, Bessereau J-L (2007) Mos1-mediated insertional mutagenesis in Caenorhabditis elegans. Nat Protoc 2:1276–1287. https://doi.org/10.1038/nprot.2007.192

    Article  CAS  PubMed  Google Scholar 

  23. Frøkjær-Jensen C, Jain N, Hansen L, Davis MW, Li Y, Zhao D, Rebora K, Millet JRM, Liu X, Kim SK, Dupuy D, Jorgensen EM, Fire AZ (2016) An abundant class of non-coding DNA can prevent stochastic gene silencing in the C. elegans germline. Cell 166:343–357. https://doi.org/10.1016/j.cell.2016.05.072

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Aljohani MD, El Mouridi S, Priyadarshini M, Vargas-Velazquez AM, Frøkjær-Jensen C (2020) Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in C. elegans. Nat Commun 11:6300. https://doi.org/10.1038/s41467-020-19898-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pokala N, Liu Q, Gordus A, Bargmann CI (2014) Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels. Proc Natl Acad Sci U S A 111:2770–2775. https://doi.org/10.1073/pnas.1400615111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. El Mouridi S, AlHarbi S, Frøkjær-Jensen C (2021) A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. MicroPubl Biol 2021. https://doi.org/10.17912/micropub.biology.000349

  27. Shirayama M, Seth M, Lee H-C, Gu W, Ishidate T, Conte D Jr, Mello CC (2012) piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell 150:65–77. https://doi.org/10.1016/j.cell.2012.06.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Research in the laboratory of CFJ is supported by KAUST intramural funding. Strains for transgene insertion are provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and by the NemaGENETAG project. We thank the Bargmann lab (Rockefeller University) for plasmids used for histamine-based selection.

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Authors and Affiliations

  1. Biological and Environmental Science and Engineering Division (BESE), KAUST Environmental Epigenetics Program (KEEP), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

    Sonia El Mouridi & Christian Frøkjær-Jensen

Authors
  1. Sonia El Mouridi
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  2. Christian Frøkjær-Jensen
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Corresponding author

Correspondence to Christian Frøkjær-Jensen .

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Editors and Affiliations

  1. Department of Biological Sciences, New Jersey Institute of Technology, Newark, NJ, USA

    Gal Haspel

  2. Department of Neuroscience, Brown University, Providence, RI, USA

    Anne C. Hart

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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El Mouridi, S., Frøkjær-Jensen, C. (2022). Targeted and Random Transposon-Assisted Single-Copy Transgene Insertion in C. elegans. In: Haspel, G., Hart, A.C. (eds) C. elegans. Methods in Molecular Biology, vol 2468. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2181-3_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2181-3_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2180-6

  • Online ISBN: 978-1-0716-2181-3

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