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.

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.