Combinatorial tumor suppressor gene targeting immortalizes primary pancreatic ductal cells, resulting in temporal, deterministic structural patterns across the genome

Felsenstein, Matthäus; Hu, Mengwen; Ma, Dou; Tamboli, Jenny; Jin, Cao Zhong Jing; Wang, Ruonan; Hui, Luan Xiao; Dubois, Frank; Neumann, Christopher; Dolnik, Anna; Hillebrandt, Karl Herbert; Haderer, Luna; Giurgiu, Madalina; Henssen, Anton; Kübler, Kirsten; Pratschke, Johann; Malinka, Thomas; Sauer, Igor; Wood, Laura; Roberts, Nicholas

Combinatorial tumor suppressor gene targeting immortalizes primary pancreatic ductal cells, resulting in temporal, deterministic structural patterns across the genome

Tools

Preview	PDF (Preprint) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader 3MB
	Other (Supplementary Files) 8MB

Item Type:	Preprint
Title:	Combinatorial tumor suppressor gene targeting immortalizes primary pancreatic ductal cells, resulting in temporal, deterministic structural patterns across the genome
Creators Name:	Felsenstein, Matthäus, Hu, Mengwen, Ma, Dou, Tamboli, Jenny, Jin, Cao Zhong Jing, Wang, Ruonan, Hui, Luan Xiao, Dubois, Frank, Neumann, Christopher, Dolnik, Anna, Hillebrandt, Karl Herbert, Haderer, Luna, Giurgiu, Madalina, Henssen, Anton, Kübler, Kirsten, Pratschke, Johann, Malinka, Thomas, Sauer, Igor, Wood, Laura and Roberts, Nicholas
Abstract:	Pancreatic carcinoma develops from pre-cancerous lesions. Limited sensitivity of current imaging modalities and biomarkers challenges early detection. Findings from murine in vivo models revealed deterministic structural patterns following heterozygous TP53 alteration. Few pancreatic duct cell culture models exist to study such molecular concepts in the early stages of the disease process. A Golden Gate assembly cloning method was used to generate multiplexed vectors that targeted the main pancreatic carcinoma driver genes (KRAS, CDKN2A, TP53, SMAD4). Their transient transfection was implemented in six experimental arms in HEK293T and primary ductal cells. Immortalized cell lines were characterized via established in vitro assays. In-depth whole exome (>30x) and nanopore sequencing (>15x) at two distinct evolutionary time points was conducted. Multiplexed vectors targeted main driver genes with an observed NHEJ (non-homologous end joining) e ciency of up to 25%. Following transient transfection of primary pancreatic ductal cells, six cell lines were generated from three patient samples, all of which exhibited loss of function in the two genes, CDKN2A and TP53. The immortalized cell lines maintained a duct cell phenotype. In-depth genomic signature analysis con rmed gene editing and revealed deterministic structural changes from early to later passages. Our study successfully demonstrated the immortalization of pancreatic duct epithelial cells through combinatorial CRISPR/Cas9 gene editing. The methodology could be expanded to other di cult-to-transfect cells. Driver gene targeting in human pancreatic ductal cells resulted in deterministic structural patterns that could explain the accelerated carcinogenesis at early stages.
Keywords:	Pancreatic Precursor Lesion, CRISPR Gene Targeting, Deterministic Structural Pattern
Source:	Research Square
Publisher:	Research Square
Article Number:	rs-9124723/v1
Date:	27 March 2026
Official Publication:	https://doi.org/10.21203/rs.3.rs-9124723/v1
Related to:	URL URL Type https://ega-archive.org/datasets/EGAD50000001842 External Dataset

Repository Staff Only: item control page

Download Statistics

Downloads

Downloads per month over past year