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


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 



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.


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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

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