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