CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines

Eva Lentsch, Lifei Li, Susanne Pfeffer, Arif B Ekici, Leila Taher, Christian Pilarsky, Robert Grützmann

Research output: Contribution to journalArticleResearchpeer-review

Abstract

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.

Original languageEnglish
Article number5706
Number of pages11
JournalInternational Journal of Molecular Sciences
Volume20
Issue number22
DOIs
Publication statusPublished - 14 Nov 2019

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CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Pancreatic Neoplasms
cultured cells
cancer
Cells
mutations
proteins
Proteins
Cell Line
cells
Signal transduction
Mutation
Signal Transduction
Clone Cells
Puromycin
AMP-Activated Protein Kinases
Proto-Oncogenes
oncogenes
Cloning

Fields of Expertise

  • Human- & Biotechnology

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CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines. / Lentsch, Eva; Li, Lifei; Pfeffer, Susanne; Ekici, Arif B; Taher, Leila; Pilarsky, Christian; Grützmann, Robert.

In: International Journal of Molecular Sciences , Vol. 20, No. 22, 5706, 14.11.2019.

Research output: Contribution to journalArticleResearchpeer-review

Lentsch, Eva ; Li, Lifei ; Pfeffer, Susanne ; Ekici, Arif B ; Taher, Leila ; Pilarsky, Christian ; Grützmann, Robert. / CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines. In: International Journal of Molecular Sciences . 2019 ; Vol. 20, No. 22.
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