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Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo

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Translational Research Methods in Neurodevelopmental Disorders

Part of the book series: Neuromethods ((NM,volume 185))

Abstract

Primary cultured neurons represent a benchmark preparation for studying neuronal cell biology. However, due to their poor transfection efficiency, studies often require virus-mediated gene delivery to genetically alter primary neurons. Lentiviral systems, based on HIV, are a commonly used gene delivery system due to their efficient expression, ease of production, and low toxicity to target cells. A powerful technique in interrogating protein function is ablation of specific proteins from cells using RNAi. In particular, expression of shRNA against the target protein allows for stable, long-term reduction of the protein of interest. Here, we describe a lentiviral system we use to knock down target proteins in primary cultured neurons via expression of an shRNA. We outline the cloning steps required to produce an shRNA-expressing lentiviral construct and provide a detailed method that can be used to produce lentiviral particles to transduce cultured neurons. In addition, we describe how to concentrate the produced lentiviral particles, allowing their use for in vivo studies.

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References

  1. Fletcher-Jones A, Hildick KL, Evans AJ et al (2019) The C-terminal helix 9 motif in rat cannabinoid receptor type 1 regulates axonal trafficking and surface expression. elife 8:e44252. https://doi.org/10.7554/eLife.44252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jaafari N, Konopacki FA, Owen TF et al (2013) SUMOylation is required for glycine-induced increases in AMPA receptor surface expression (ChemLTP) in hippocampal neurons. PLoS One 8:e52345. https://doi.org/10.1371/journal.pone.0052345

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chamberlain SEL, González-González IM, Wilkinson KA et al (2012) SUMOylation and phosphorylation of GluK2 regulate kainate receptor trafficking and synaptic plasticity. Nat Neurosci 15:845–852. https://doi.org/10.1038/nn.3089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Konopacki FA, Jaafari N, Rocca DL et al (2011) Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis. Proc Natl Acad Sci U S A 108:19772–19777. https://doi.org/10.1073/pnas.1111575108

    Article  PubMed  PubMed Central  Google Scholar 

  5. Carmichael RE, Wilkinson KA, Craig TJ et al (2018) MEF2A regulates mGluR-dependent AMPA receptor trafficking independently of Arc/Arg3.1. Sci Rep 8:5263. https://doi.org/10.1038/s41598-018-23440-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gurung S, Evans AJ, Wilkinson KA, Henley JM (2018) ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity. J Cell Sci 131. https://doi.org/10.1242/jcs.222273

  7. Evans AJ, Gurung S, Wilkinson KA et al (2017) Assembly, secretory pathway trafficking, and surface delivery of kainate receptors is regulated by neuronal activity. Cell Rep 19:2613–2626. https://doi.org/10.1016/j.celrep.2017.06.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rocca DL, Wilkinson KA, Henley JM (2017) SUMOylation of FOXP1 regulates transcriptional repression via CtBP1 to drive dendritic morphogenesis. Sci Rep 7:877. https://doi.org/10.1038/s41598-017-00707-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ding B, Kilpatrick DL (2013) Lentiviral vector production, titration, and transduction of primary neurons. Methods Mol Biol 1018:119–131. https://doi.org/10.1007/978-1-62703-444-9_12

    Article  CAS  PubMed  Google Scholar 

  10. Bellizzi A, Ahye N, Wollebo HS (2021) Lentiviral transduction of neuronal cells. Methods Mol Biol 2311:155–160. https://doi.org/10.1007/978-1-0716-1437-2_11

    Article  CAS  PubMed  Google Scholar 

  11. Li M, Husic N, Lin Y, Snider BJ (2012) Production of lentiviral vectors for transducing cells from the central nervous system. J Vis Exp 63:e4031. https://doi.org/10.3791/4031

    Article  CAS  Google Scholar 

  12. Sioud M (2021) RNA interference: story and mechanisms. Methods Mol Biol 2282:1–15. https://doi.org/10.1007/978-1-0716-1298-9_1

    Article  CAS  PubMed  Google Scholar 

  13. Elbashir SM, Harborth J, Lendeckel W et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498. https://doi.org/10.1038/35078107

    Article  CAS  PubMed  Google Scholar 

  14. Caplen NJ, Parrish S, Imani F et al (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A 98:9742–9747. https://doi.org/10.1073/pnas.171251798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239. https://doi.org/10.1146/annurev-biophys-083012-130404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553. https://doi.org/10.1126/science.1068999

    Article  CAS  PubMed  Google Scholar 

  17. Sui G, Soohoo C, Affar EB et al (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A 99:5515–5520. https://doi.org/10.1073/pnas.082117599

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508. https://doi.org/10.1038/nbt0502-505

    Article  CAS  PubMed  Google Scholar 

  19. Yu J-Y, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99:6047–6052. https://doi.org/10.1073/pnas.092143499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524. https://doi.org/10.1038/nrm3838

    Article  CAS  PubMed  Google Scholar 

  21. Nakamura Y, Morrow DH, Nathanson AJ et al (2020) Phosphorylation on Ser-359 of the α2 subunit in GABA type A receptors down-regulates their density at inhibitory synapses. J Biol Chem 295:12330–12342. https://doi.org/10.1074/jbc.RA120.014303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi VH, Craig TJ, Bishop P et al (2021) Phosphorylation of Syntaxin-1a by casein kinase 2α regulates pre-synaptic vesicle exocytosis from the reserve pool. J Neurochem 156:614–623. https://doi.org/10.1111/jnc.15161

    Article  CAS  PubMed  Google Scholar 

  23. Binda CS, Nakamura Y, Henley JM, Wilkinson KA (2019) Sorting nexin 27 rescues neuroligin 2 from lysosomal degradation to control inhibitory synapse number. Biochem J 476:293–306. https://doi.org/10.1042/BCJ20180504

    Article  CAS  PubMed  Google Scholar 

  24. Demaison C, Parsley K, Brouns G et al (2002) High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 13:803–813. https://doi.org/10.1089/10430340252898984

    Article  CAS  PubMed  Google Scholar 

  25. Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73:2886–2892. https://doi.org/10.1128/JVI.73.4.2886-2892.1999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McMillan KJ, Banks PJ, Hellel FL et al (2021) Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2. elife 10:e59432. https://doi.org/10.7554/eLife.59432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. (2003) Whither RNAi? Nat Cell Biol 5:489–490

    Google Scholar 

  28. Choy RW-Y, Park M, Temkin P et al (2014) Retromer mediates a discrete route of local membrane delivery to dendrites. Neuron 82:55–62. https://doi.org/10.1016/j.neuron.2014.02.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kim Y-M, Stone M, Hwang TH et al (2012) SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling. Mol Cell 46:833–846. https://doi.org/10.1016/j.molcel.2012.04.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307:1098–1101. https://doi.org/10.1126/science.1106148

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank the BBSRC (BB/R00787X/1 to KAW and JMH), the Leverhulme Trust (RPG-2019-191 to JMH), and a Wellcome Trust Investigatorship (220799/Z/20/Z) to JMH for funding this work.

Author information

Authors and Affiliations

  1. School of Biochemistry, University of Bristol, Bristol, UK

    Kevin A. Wilkinson, Kirsty J. McMillan, Yasuko Nakamura, Peter J. Cullen & Jeremy M. Henley

  2. School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK

    Paul J. Banks & Zafar I. Bashir

  3. Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK

    Ruth E. Carmichael

  4. Centre for Neuroscience and Regenerative Medicine, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia

    Jeremy M. Henley

Authors
  1. Kevin A. Wilkinson
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  2. Kirsty J. McMillan
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  3. Paul J. Banks
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  4. Ruth E. Carmichael
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  5. Yasuko Nakamura
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  6. Zafar I. Bashir
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  7. Peter J. Cullen
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  8. Jeremy M. Henley
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Corresponding author

Correspondence to Kevin A. Wilkinson .

Editor information

Editors and Affiliations

  1. CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France

    Stéphane Martin

  2. UMR1253, iBrain, University of Tours, Inserm, Tours, France

    Frédéric Laumonnier

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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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Wilkinson, K.A. et al. (2022). Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo. In: Martin, S., Laumonnier, F. (eds) Translational Research Methods in Neurodevelopmental Disorders. Neuromethods, vol 185. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2569-9_1

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  • DOI: https://doi.org/10.1007/978-1-0716-2569-9_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2568-2

  • Online ISBN: 978-1-0716-2569-9

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