CRISPR/Cas9-mediated knock-out of krasG12D mutated pancreatic cancer cell lines

Lentsch E, Li L, Pfeffer S, Ekici AB, Taher L, Pilarsky C, Grützmann R (2019)

Publication Type: Journal article

Publication year: 2019

Journal

International Journal of Molecular Sciences MDPI

Book Volume: 20

Article Number: 5706

Journal Issue: 22

DOI: 10.3390/ijms20225706

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.

Authors with CRIS profile

Lifei Li Juniorprofessur für Bioinformatik Sina Pfeffer Medizinische Fakultät Arif Bülent Ekici Institute of Human Genetics Leila Taher Juniorprofessur für Bioinformatik Christian Pilarsky Professur für Chirurgische Forschung Robert Grützmann Lehrstuhl für Allgemein- und Viszeralchirurgie

How to cite

APA:

Lentsch, E., Li, L., Pfeffer, S., Ekici, A.B., Taher, L., Pilarsky, C., & Grützmann, R. (2019). CRISPR/Cas9-mediated knock-out of krasG12D mutated pancreatic cancer cell lines. International Journal of Molecular Sciences, 20(22). https://doi.org/10.3390/ijms20225706

MLA:

Lentsch, Eva, et al. "CRISPR/Cas9-mediated knock-out of krasG12D mutated pancreatic cancer cell lines." International Journal of Molecular Sciences 20.22 (2019).

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