A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Investigating the involvement of the ATF6α pathway of the unfolded protein response in adipogenesis.

The unfolded protein response (UPR) is activated by endoplasmic reticulum stress resulting from an accumulation of unfolded or mis-folded proteins. The UPR is divided into three arms, involving the activation of ATF-6, PERK and IRE-1, that together act to restrict new protein synthesis and increase the production of chaperones. Recent studies have implicated the PERK and IRE-1 components of the UPR in adipocyte differentiation. In this study, we investigate the importance of ATF6α during adipogenesis using stable knockdown of this protein in the model adipogenic cell line, C3H10T1/2. Reduction of ATF6α expression by >70% resulted in impaired expression of key adipogenic genes and reduced lipid accumulation following the induction of adipogenesis. In contrast, loss of ATF6α did not impair the ability of cells to undergo osteogenic differentiation. Overall, our data indicate that all three arms of the UPR, including ATF6α, must be intact to permit adipogenesis to occur.

Opportunity costs of gastrointestinal endoscopic training in Canada.

BACKGROUND: No data exist to define the opportunity costs related to instruction in endoscopic procedures in Royal College of Physicians and Surgeons of Canada-accredited teaching centres. Academic and institutional administrators expect staff to achieve acceptable performance standards. There is a need to measure some of the effects of training activity in the establishment of such standards. OBJECTIVE: To measure the effect of resident training in colonoscopy on real procedure times and, as a secondary goal, to estimate procedural losses related to the process of training. METHODS: Real procedure times for ambulatory colonoscopy in a single academic, hospital-based endoscopy unit were documented. Times for certified endoscopy instructors functioning solo were compared with times for procedures involving trainees at several levels of colonoscopic experience. Procedural reductions associated with resident training were estimated based on the parameters derived from the results. The analysis was executed retrospectively using prospectively collected data. RESULTS: Resident training prolonged procedure times for ambulatory colonoscopy by 50%. The trainee effect was consistent, although variable in degree, among a variety of endoscopy instructors. Such increased procedure times have the potential to reduce case throughput and endoscopist remuneration. CONCLUSIONS: Resident training in colonoscopy in a Canadian certified training program has significant negative effects on case throughput and endoscopist billings. These factors should be considered in any assessment of performance in similar training environments.

Association analysis of the IGF1 gene with childhood growth, IGF-1 concentrations and type 1 diabetes.

AIMS/HYPOTHESIS: Insulin-like growth factor-1 is a major childhood growth factor and promotes pancreatic islet cell survival and growth in vitro. We hypothesised that genetic variation in IGF1 might be associated with childhood growth, glucose metabolism and type 1 diabetes risk. We therefore examined the association between common genetic variation in IGF1 and predisposition to type 1 diabetes, childhood growth and metabolism. MATERIALS AND METHODS: Variants in IGF1 were identified by direct resequencing of the exons, exon-intron boundaries and 5' and 3' regions in 32 unrelated type 1 diabetes patients. A tagging subset of these variants was genotyped in a collection of type 1 diabetes families (3,121 parent-child trios). We also genotyped a previously reported CA repeat in the region 5' to IGF1. A subset of seven tag single nucleotide polymorphism (SNPs) that captured variants with minor allele frequency (MAF) > or =0.05 was genotyped in 902 children from the Avon Longitudinal Study of Parents And Children with data on growth, IGF-1 concentrations, insulin secretion and insulin action. RESULTS: Resequencing detected 27 SNPs in IGF1, of which 11 had a MAF > 0.05 and were novel. Variants with MAF > or = 0.10 were captured by a set of four tag-SNPs. These SNPs showed no association with type 1 diabetes. In children, global variation in IGF1 was weakly associated with IGF-1 concentrations, but not with other phenotypes. The CA repeat in the region 5' to IGF1 showed no association with any phenotype. CONCLUSIONS/INTERPRETATION: Common genetic variation in IGF1 alters IGF-1 concentrations but is not associated with growth, glucose metabolism or type 1 diabetes.

Upper gastrointestinal toxicity of alendronate.

OBJECTIVE: Alendronate is rapidly gaining widespread use in the treatment of osteoporosis. However, recent postmarketing surveys and endoscopic studies suggest that its use may be associated with significant predictable esophageal and gastric mucosal toxicity, similar to that of aspirin and nonsteroidal anti-inflammatory drugs. Because treatment of osteoporosis may be needed in as many as 30% of all postmenopausal women, and considering that alendronate could be used in all postmenopausal women as prevention, definition of potential mucosal toxicity is crucial. Our aim was to study the upper gastrointestinal toxicity of alendronate in an age-appropriate female population using a clinically applicable dose (10 mg/day) to determine whether it causes predictable damage in the proximal gastrointestinal mucosa in a fashion similar to that seen with aspirin and nonsteroidal anti-inflammatory drugs. METHODS: We conducted a double-blind, randomized, placebo-controlled trial in 32 healthy female volunteers between the ages of 40 and 65 yr recruited by newspaper advertisement. Endoscopic mucosal abnormalities in the esophagus, stomach, and duodenum both before and after 1 month of treatment were scored and compared using validated endoscopic grading systems. Symptom scores before and after treatment were determined. Noninvasive measurements of gastrointestinal permeability were obtained before, during, and after treatment using sucrose and mannitol/lactulose urinary excretions. RESULTS: Endoscopic scores before and after treatment with alendronate were not significantly different. Similarly, mean symptom scores in the alendronate group did not change significantly after treatment. There were no significant mucosal permeability changes in the stomach or small intestine after treatment. CONCLUSION: Alendronate does not cause predictable esophageal, gastric, or duodenal mucosal damage when used as directed.

Cost-effective analysis of candidate genes using htSNPs: a staged approach.

We have previously shown that the selection of haplotype tag single nucleotide polymorphisms (htSNPs) and their statistical analysis in a multi-locus transmission/disequilibrium test (TDT) results in a more cost-effective genotyping strategy in disease association studies of genes by minimising redundancy due to linkage disequilibrium between SNPs. Further savings can be achieved by the use of a two-stage genotyping strategy. This approach is illustrated here in conjunction with the multi-locus TDT in determining whether common alleles of the immune regulatory genes RANK and its ligand TRANCE (RANKL) are associated with type 1 diabetes (T1D). A saving of approximately 75% of potential genotyping reactions could be made with minimal loss of power. There was little evidence from our analysis for association between the TRANCE and RANK genes and T1D in the populations tested.