Advertisement

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

  • Khanti Rattanapornsompong
  • Jarunya Ngamkham
  • Tanit Chavalit
  • Sarawut JitrapakdeeEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Pyruvate carboxylase Metabolism Cancer Short hairpin RNA CRISPR Cas9 Knockout 

Notes

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.

References

  1. 1.
    Jitrapakdee S, St Maurice M, Rayment I, Cleland WW, Wallace JC, Attwood PV (2008) Structure, mechanism and regulation of pyruvate carboxylase. Biochem J 413:369–387CrossRefGoogle Scholar
  2. 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 283:28048–28059CrossRefGoogle Scholar
  3. 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 108:8674–8679CrossRefGoogle Scholar
  4. 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:e0129848CrossRefGoogle Scholar
  5. 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 125:687–698CrossRefGoogle Scholar
  6. 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 17:1317–1326CrossRefGoogle Scholar
  7. 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. 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 23:517–528CrossRefGoogle Scholar
  9. 9.
    Eisener-Dorman AF, Lawrence DA, Boliva VJ (2009) Cautionary insights on knockout mouse studies: the gene or not the gene? Brain Behav Immun 23:318–324CrossRefGoogle Scholar
  10. 10.
    Bouabe H, Okkenhaug K (2013) Gene targeting in mice: a review. Methods Mol Biol 1064:315–336CrossRefGoogle Scholar
  11. 11.
    Marin-Valencia I, Roe CR, Pascual JM (2010) Pyruvate carboxylase deficiency: mechanisms, mimics and anaplerosis. Mol Genet Metab 101:9–17CrossRefGoogle Scholar
  12. 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 62:2183–2194CrossRefGoogle Scholar
  13. 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 1863:537–555CrossRefGoogle Scholar
  14. 14.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefGoogle Scholar
  15. 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 20:4903–4912CrossRefGoogle Scholar
  16. 16.
    Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, N.YGoogle Scholar
  17. 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 25:402–408CrossRefGoogle Scholar
  18. 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 76:4350–4354CrossRefGoogle Scholar
  19. 19.
    Chandler CS, Ballard FJ (1986) Multiple biotin-containing proteins in 3T3-L1 cells. Biochem J 237:123–130CrossRefGoogle Scholar
  20. 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 90:8392–8396CrossRefGoogle Scholar
  21. 21.
    Shah SA, Erdmann S, Mojica FJ, Garrett RA (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol 10:891–899CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Khanti Rattanapornsompong
    • 1
  • Jarunya Ngamkham
    • 2
  • Tanit Chavalit
    • 1
  • Sarawut Jitrapakdee
    • 1
    Email author
  1. 1.Department of Biochemistry, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Research DivisionNational Cancer InstituteBangkokThailand

Personalised recommendations