| Chapter title |
CRISPR/Cas9-Mediated Generation of Niemann–Pick C1 Knockout Cell Line
|
|---|---|
| Chapter number | 7 |
| Book title |
Cholesterol Homeostasis
|
| Published in |
Methods in molecular biology, February 2017
|
| DOI | 10.1007/978-1-4939-6875-6_7 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6873-2, 978-1-4939-6875-6
|
| Authors |
Ximing Du, Ivan Lukmantara, Hongyuan Yang, Du, Ximing, Lukmantara, Ivan, Yang, Hongyuan |
| Editors |
Ingrid C. Gelissen, Andrew J. Brown |
| Abstract |
Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene. The protocol details the steps for single guide RNA oligo cloning, cell colony selection, and cell line verification by filipin staining and immunoblotting. |
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Mendeley readers
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