Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting intronic sequences flanked by two loxP sites within an exon of a gene using CRISPR-Cas9 technology. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translational termination. Here we describe the application of this technology to engineer conditional alleles in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Deletion of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.
Abstract Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting intronic sequences flanked by two loxP sites within an exon of a gene using CRISPR-Cas9 technology. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translational termination. Here we describe the application of this technology to engineer conditional alleles in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Deletion of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.
Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.
Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats, and an overall disorganized C-terminal segment. In mammals, three family members encoded by genes Scyl1 , Scyl2 , and Scyl3 have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3. Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack of Scyl3 in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused by Scyl1 deficiency. Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves occurred at an earlier age in Scyl1 /S cyl3 double-deficient mice than in Scyl1 -deficient mice. Disease onset also correlated with the mislocalization of TDP-43 in spinal motor neurons, suggesting that SCYL1 and SCYL3 regulate TDP-43 proteostasis. Together, our results demonstrate an overlapping role for SCYL1 and SCYL3 in vivo and highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study. SIGNIFICANCE STATEMENT SCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption of Scyl3 in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss of Scyl1 , a novel motor neuron disease gene in humans. Scyl1 and Scyl3 double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than did Scyl1 -deficient mice. Thus, we show that SCYL1 and SCYL3 play overlapping roles in maintaining motor neuronal viability in vivo and confirm that SCYL family members are critical regulators of neuronal function and survival.
Abstract Invasive pancreatic adenocarcinoma (PDAC) is one of the most lethal solid malignancies. Mutational activation of Kras is nearly universal in preneoplastic pancreatic lesions (Pancreatic Intraepithelial Neoplasias-PanINs) and PDAC, whereas the progression of PanINs to PDAC is accompanied by both inactivating mutations in p16/CDKN2A, TP53, DPC4/SMAD4 and activating mutations in PI3K signaling. Although most PDAC cases represent a sporadic disease, about 5-10% of cases have a hereditary basis. Some of the loci responsible for inherited high-risk PDAC encode gene products implicated in DNA repair, such as BRCA2 and PALB2. The kinase Ataxia-Telangiectasia Mutated (ATM) plays a critical role in DNA damage responses activated by double-strand breaks, by triggering cell cycle checkpoints that repair DNA or induce p53-dependent apoptosis or senescence. ATM was recently identified as a novel predisposition gene for PDAC because deleterious ATM mutations were found in some families with hereditary PDAC, as well as in ~8% of sporadic PDAC cases. Thus far, the role of ATM as an anti-cancer barrier – especially in pancreatic cancer biology – is poorly understood. Here we sought to address this crucial issue using 3 different mouse strains expressing both an oncogenic version of Kras (KrasG12D) and variable ATM genetic dosage in the pancreas: 1) KC mice, express wild-type ATM, 2) KC;ATMcHET mice, carry deletion of one ATM-allele; and 3) KC;ATMcKO mice, carry deletion of both ATM-alleles. Our preliminary analysis of KrasG12D-induced PanIN formation uncovered that these lesions form earlier in pancreatic tissues carrying one functional ATM allele or lacking ATM in comparison to KC pancreata. In addition, we determined that the median survival due to metastatic tumor burden is considerably shorter in both KC;ATMcKO mice (~6 months) and KC;ATMcHET mice (~9 months) compared to KC mice (~15 months). We also began establishing primary cell lines from both PDAC and metastatic foci formed in KC, KC;ATMcKO and KC;ATMcHET mice. Preliminary analyses of 2 primary cell lines treated with ionizing radiation (IR) to induce DNA damage and processed for Western blot analysis showed lack of total ATM, phospho-ATM, phospho-KAP1 (an ATM-specific target), and phospho-p53 in ATM-deficient primary pancreatic cancer cells. In contrast, primary cell lines from KC;Cdkn2a+/- tumors and established human PDAC cell lines display functional ATM signaling after IR. In addition, primary cell lines from PDAC and liver metastases of KC;ATMcKO mice also exhibited genomic instability depicted by a high number of anaphase bridges. To complement the characterization of these primary cell lines, we will attempt to identify DNA repair pathway(s) compensating for the loss of ATM and exploit these findings to induce synthetic lethality. Likewise, additional signaling pathways that are activated or inactivated during PDAC progression in the context of ATM deficiency will be identified using both biased and unbiased approaches. To our knowledge, these preliminary results provide the first evidence in vivo that ATM activity poses an intrinsic barrier to oncogenic transformation in the pancreas. A comprehensive characterization of pancreatic tumor formation in the context of ATM deficiency using our generated mouse models is currently underway, together with experiments in primary cell lines attempting to identify vulnerabilities that could be exploited to develop adequate tailored treatments. Citation Format: Yiannis Drosos, Kathryn Roys, Emin Kuliyev, Peter McKinnon, Beatriz Sosa-Pineda. ATM loss and KrasG12D cooperate to promote widely metastatic pancreatic ductal adenocarcinoma in mice. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A06.
