Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Background & Aims The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aim to evaluate the role of PTEN in the pathogenesis of eCCA and find novel therapies for this disease. Methods The Pten gene in the biliary epithelial cells were genetically deleted using the Cre-loxp system. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry (IHC), RT-PCR, cell culture, and RNAseq. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. Experimental therapy was tested using an Aurka inhibitor. Results We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as one month old. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated the disease progression, potentially through downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expressions of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. Conclusions Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum through an Aurka-dependent manner. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. Impact and implications The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition (EMT), cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted the disease progression. This model shall be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

Background: Tissue and cell-specific gene targeting has been widely employed in biomedical research. In the pancreas, the commonly used Cre recombinase recognizes and recombines loxP sites. However, to selectively target different genes in distinct cells, a dual recombinase system is required.Method: We developed an alternative recombination system mediated by FLPo, which recognizes frt DNA sequences for pancreatic dual recombinase-mediated genetic manipulation. An IRES-FLPo cassette was targeted between the translation stop code and 3-UTR of the mouse pdx1 gene in a Bacterial Artificial Chromosome using recombineering technology. Transgenic BAC-Pdx1-FLPo mice were developed by pronuclear injection.Results: Highly efficient recombination activity was observed in the pancreas by crossing the founder mice with Flp reporter mice. When the BAC-Pdx1-FLPo mice were bred with conditional FSF-KRasG12D and p53 F/F mice, pancreatic cancer developed in the compound mice. The characteristics of pancreatic cancer resembled those derived from conditional LSL-KRasG12D and p53 L/L mice controlled by pdx1-Cre.Conclusions: We have generated a new transgenic mouse line expressing FLPo, which enables highly efficient pancreatic-specific gene recombination. When combined with other available Cre lines, this system can be utilized to target different genes in distinct cells for pancreatic research.(c) 2023 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Premature digestive enzyme activation in the pancreas is a major cause of pancreatitis. This concept is supported by the observation that intrapancreatic trypsinogen activation is one of the earliest events in experimental pancreatitis models. 1 Saluja A. et al. Gastroenterology. 2019; 156: 1979-1993 Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Genetic expression of "super-active" murine trypsin mutants induces pancreatitis. 2 Gaiser S. et al. Gut. 2011; 60: 1379-1388 Crossref PubMed Scopus (85) Google Scholar ,3 Geisz A. et al. Nat Commun. 2018; 9: 5033 Crossref PubMed Scopus (44) Google Scholar Most importantly, the gain-of-function trypsinogen PRSS1 gene mutations are linked to the development of human hereditary pancreatitis with >80% penetrance, a significant risk factor for pancreatic cancer. 4 Whitcomb D.C. et al. Nat Genet. 1996; 14: 141-145 Crossref PubMed Scopus (1329) Google Scholar ,5 Szabo A. et al. J Biol Chem. 2012; 287: 20701-20710 Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar However, since the discovery 25 years ago of PRSS1R122H—the most common mutation in human hereditary pancreatitis 4 Whitcomb D.C. et al. Nat Genet. 1996; 14: 141-145 Crossref PubMed Scopus (1329) Google Scholar —the many attempts to develop a genetic mouse model for hereditary pancreatitis by transgenic expression of human PRSS1R122H have yielded limited success. 6 Athwal T. et al. Cell Death Dis. 2014; 5: e1165 Crossref PubMed Scopus (27) Google Scholar , 7 Gui F. et al. J Clin Invest. 2020; 130: 189-202 Crossref PubMed Scopus (30) Google Scholar , 8 Huang H. et al. Gastroenterology. 2020; 158: 1072-1082.e7 Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Some of these transgenic models can substantially worsen cerulein-indued pancreatitis; however, none of these models showed evidence of spontaneous pancreatitis. 6 Athwal T. et al. Cell Death Dis. 2014; 5: e1165 Crossref PubMed Scopus (27) Google Scholar , 7 Gui F. et al. J Clin Invest. 2020; 130: 189-202 Crossref PubMed Scopus (30) Google Scholar , 8 Huang H. et al. Gastroenterology. 2020; 158: 1072-1082.e7 Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar The underlying reasons are unclear.