Skip to main content
SpringerLink
Log in

Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research

  • Protocol
  • First Online:
Pre-Clinical Models

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1916))

Abstract

We report two protocols to generate human pyruvate carboxylase knockdown and knockout cell lines using short hairpin RNA (shRNA) and CRISPR-Cas9 technologies. The first protocol involved cloning of a shRNA cassette targeted to human pyruvate carboxylase (PC) under the control of a U6 promoter in a retrovirus-based vector. The stable knockdown cells were achieved following infection of retroviruses expressing shRNA in target cells followed by selecting these in medium containing puromycin. The second protocol describes a CRISPR Cas9-knockout cell constructed by cloning of single guide RNA (gRNA) targeted to the human pyruvate carboxylase gene placed adjacent to Cas 9 in the pSpCas9(BB)-2A-GFP vector. The knockout cells can be selected by sorting the cells expressing GFP. We also describe protocols for detecting the level of PC mRNA and protein in the knockdown or knockout cells using qPCR and Western blot analyses, respectively. The above protocols allow investigators to create PC deficient cell lines as a tool to study role of this enzyme in cancer research.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
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. Jitrapakdee S, St Maurice M, Rayment I, Cleland WW, Wallace JC, Attwood PV (2008) Structure, mechanism and regulation of pyruvate carboxylase. Biochem J 413:369–387

    Article  CAS  Google Scholar 

  2. Hasan NM, Longacre MJ, Stoker SW, Boonsaen T, Jitrapakdee S, Kendrick MA et al (2008) Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase. J Biol Chem 283:28048–28059

    Article  CAS  Google Scholar 

  3. Cheng T, Sudderth J, Yang C, Mullen AR, Jin ES, Matés JM et al (2011) Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proc Natl Acad Sci U S A 108:8674–8679

    Article  CAS  Google Scholar 

  4. Phannasil P, Thuwajit C, Warnnissorn M, Wallace JC, MacDonald MJ, Jitrapakdee S (2015) Pyruvate carboxylase is up-regulated in breast cancer and essential to support growth and invasion of MDA-MB-231 cells. PLoS One 10:e0129848

    Article  Google Scholar 

  5. Sellers K, Fox MP, Bousamra M, Slone SP, Higashi RM, Miller DM et al (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J Clin Invest 125:687–698

    Article  Google Scholar 

  6. Cardaci S, Zheng L, MacKay G, van den Broek NJ, MacKenzie ED, Nixon C et al (2015) Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis. Nat Cell Biol 17:1317–1326

    Article  CAS  Google Scholar 

  7. Lussey-Lepoutre C, Hollinshead KE, Ludwig C, Menara M, Morin A, Castro-Vega LJ et al (2015) Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism. Nat Commun 6:8784. https://doi.org/10.1038/ncomms9784

  8. Davidson SM, Papagiannakopoulos T, Olenchock BA, Heyman JE, Keibler MA, Luengo A et al (2016) Environment impacts the metabolic dependencies of Ras-driven non-small cell lung cancer. Cell Metab 23:517–528

    Article  CAS  Google Scholar 

  9. Eisener-Dorman AF, Lawrence DA, Boliva VJ (2009) Cautionary insights on knockout mouse studies: the gene or not the gene? Brain Behav Immun 23:318–324

    Article  CAS  Google Scholar 

  10. Bouabe H, Okkenhaug K (2013) Gene targeting in mice: a review. Methods Mol Biol 1064:315–336

    Article  CAS  Google Scholar 

  11. Marin-Valencia I, Roe CR, Pascual JM (2010) Pyruvate carboxylase deficiency: mechanisms, mimics and anaplerosis. Mol Genet Metab 101:9–17

    Article  CAS  Google Scholar 

  12. Kumashiro N, Beddow SA, Vatner DF, Majumdar SK, Cantley JL, Guebre-Egziabher F et al (2013) Targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance. Diabetes 62:2183–2194

    Article  CAS  Google Scholar 

  13. Phannasil P, Ansari IH, El Azzouny M, Longacre MJ, Rattanapornsompong K, Burant CF et al (2017) Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells. Biochim Biophys Acta 1863:537–555

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Goomer RS, Kunkel GR (1992) The transcriptional start site for a human U6 small nuclear RNA gene is dictated by a compound promoter element consisting of the PSE and the TATA box. Nucleic Acids Res 20:4903–4912

    Article  CAS  Google Scholar 

  16. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, N.Y

    Google Scholar 

  17. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  Google Scholar 

  18. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  CAS  Google Scholar 

  19. Chandler CS, Ballard FJ (1986) Multiple biotin-containing proteins in 3T3-L1 cells. Biochem J 237:123–130

    Article  CAS  Google Scholar 

  20. Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90:8392–8396

    Article  CAS  Google Scholar 

  21. Shah SA, Erdmann S, Mojica FJ, Garrett RA (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol 10:891–899

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the International Research Network grant IRN59W003 from the Thailand Research Fund to S.J. K.R. was supported by the Science Achievement Scholarship of Thailand. We thank Professor John Wallace, University of Adelaide for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand

    Khanti Rattanapornsompong, Tanit Chavalit & Sarawut Jitrapakdee

  2. Research Division, National Cancer Institute, Bangkok, Thailand

    Jarunya Ngamkham

Authors
  1. Khanti Rattanapornsompong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jarunya Ngamkham
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tanit Chavalit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sarawut Jitrapakdee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarawut Jitrapakdee .

Editor information

Editors and Affiliations

  1. Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil

    Paul C. Guest

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Rattanapornsompong, K., Ngamkham, J., Chavalit, T., Jitrapakdee, S. (2019). Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research. In: Guest, P. (eds) Pre-Clinical Models. Methods in Molecular Biology, vol 1916. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8994-2_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8994-2_26

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8993-5

  • Online ISBN: 978-1-4939-8994-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation