Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones

By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously. To ensure inclusion of all such elements and provide details of subcellular localization, construction of translational reporters would, preferably, utilize genomic clones, containing the complete locus plus flanking regions and permit seamless insertion of the reporter anywhere within the gene. We have developed such a method based upon λ Red-mediated recombineering coupled to a robust two-step counter-selection protocol. We have inserted either gfp or cfp precisely at the C-termini of three Caenorhabditis elegans target genes, each located within different fosmid clones, and examined previously with conventional reporter approaches. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporters highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox

P155 Natural history of anal stricture in pediatric-onset Crohn’s disease: a two-decades population-based study

International audienceBackground The natural history of anal stricture complicating pediatric-onset Crohn’s disease (CD) is poorly known. The aims of this study were: to determine in a population-based study the risk of anal stricture in pediatric-onset CD; to identify risk factors for anal stricture; and to evaluate the natural history of anal stricture at the population level. Methods All patients with a diagnosis of CD made before the age of 17 years between 1988 and 2011 in a population-based registry were followed retrospectively until 2013. A specific collection of additional data was performed in patients with anal stricture at diagnosis or during follow-up. Variables collected included: proctological examination, diagnostic management and treatment. Multivariate Cox models were used to identify factors associated with anal stricture. Results Among the 1,007 included CD patients (females, 451 (44.8%); median age at diagnosis 14.4 years (IQR, 12.0-16.1), median follow-up 8.8 years (IQR, 4.6-14.2)), only one (0.1%) had anal stricture at diagnosis while 26 (2.6%) presented anal stricture during follow-up. Cumulative incidence of anal stricture at 5 and 10 years after diagnosis was 0.6% (CI95%, 0.1-1.1) and 1.4% (CI95%, 0.5-2.3), respectively. Among the 27 patients with anal stricture, 25 (92.6%) had already at least one episode of anal ulceration or fistulizing perianal CD (pCD). In multivariable analysis, the presence of extra-intestinal manifestations (Hazard Ratio (HR) 2.2, CI95% 1.0-4.8, p=0.027), colonic location (L1 vs L3 HR 0.0 (-); L2 vs L3 HR 1.2, CI95% 0.6-2.7, p=0.006) and history of fistulizing pCD (HR 9.9, CI95% 4.3-22.8, p<0.0001) were significantly associated with anal stricture. Eleven (41%) patients needed at least one dilation. After a median follow-up of 16.9 years (IQR, 11.5-20.2), healing of anal stricture was observed only once. One patient (3.7%) presented an anal cancer 7 years after the diagnosis of anal stricture. Nine (33.3%) patients needed a stoma. Among them, 8 already had a fistulizing pCD and 4 needed an abdominoperineal amputation. Anal stricture was significantly associated with an increased risk of stoma (HR 5.8, CI95% 2.3-14.3), p=0.0002). Conclusion Anal stricture is a rare event in pediatric onset CD, occurring in 1.5% of patients over a 10 year-period after diagnosis. However, anal stricture had a negative impact on disease prognosis with a five-fold increase of the risk of stoma

P1266 : Human albumin use in cirrhotic patients in France: Results of the national “Albu-live” survey

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