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

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

Full description

Saved in:
Bibliographic Details
Main Authors: Khanti Rattanapornsompong, Jarunya Ngamkham, Tanit Chavalit, Sarawut Jitrapakdee
Other Authors: National Cancer Institute Thailand
Format: Chapter
Published: 2020
Subjects:
Online Access:https://repository.li.mahidol.ac.th/handle/123456789/50318
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Mahidol University
id th-mahidol.50318
record_format dspace
spelling th-mahidol.503182020-01-27T14:53:04Z 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 Jitrapakdee National Cancer Institute Thailand Mahidol University Biochemistry, Genetics and Molecular Biology © 2019, Springer Science+Business Media, LLC, part of Springer Nature. 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. 2020-01-27T07:53:04Z 2020-01-27T07:53:04Z 2019-01-01 Chapter Methods in Molecular Biology. Vol.1916, (2019), 273-288 10.1007/978-1-4939-8994-2_26 10643745 2-s2.0-85058608941 https://repository.li.mahidol.ac.th/handle/123456789/50318 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85058608941&origin=inward
institution Mahidol University
building Mahidol University Library
continent Asia
country Thailand
Thailand
content_provider Mahidol University Library
collection Mahidol University Institutional Repository
topic Biochemistry, Genetics and Molecular Biology
spellingShingle Biochemistry, Genetics and Molecular Biology
Khanti Rattanapornsompong
Jarunya Ngamkham
Tanit Chavalit
Sarawut Jitrapakdee
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
description © 2019, Springer Science+Business Media, LLC, part of Springer Nature. 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.
author2 National Cancer Institute Thailand
author_facet National Cancer Institute Thailand
Khanti Rattanapornsompong
Jarunya Ngamkham
Tanit Chavalit
Sarawut Jitrapakdee
format Chapter
author Khanti Rattanapornsompong
Jarunya Ngamkham
Tanit Chavalit
Sarawut Jitrapakdee
author_sort Khanti Rattanapornsompong
title 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
title_short 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_sort 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
publishDate 2020
url https://repository.li.mahidol.ac.th/handle/123456789/50318
_version_ 1763494971491483648