High-throughput discovery of genetic determinants of circadian misalignment.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Characterization and utilization of chicken {\it Msx-2\/} gene regulatory sequences in the developing limb of transgenic mice

Msx-2 is a homeodomain-containing transcription factor expressed in the apical ectodermal ridge (AER) and the mesoderm at the anterior and posterior boundaries of the progress zone of the developing limb. Separate regulatory elements in the Msx-2 gene have been postulated to achieve specific spatial domains. Analysis of an 11.5kb chicken Msx-2 clone in transgenic mice has lead to the identification of a 348bp HpaII fragment 5\sp\prime of the Msx-2 promoter which is capable of driving AER expression. In the present work we have identified an additional 380bp BglII-HpaII fragment 5\sp\prime of the previously identified 348bp region which is capable of driving consistent reporter expression in the AER of the forelimbs.^ Since elements in the 11.5kb genomic clone did not drive expression in the limb mesoderm of transgenic mice, regions extending further 5\sp\prime and 3\sp\prime of the original clone were isolated. LacZ reporter constructs with chicken Msx-2 genomic sequences were generated and tested in transgenic mice to identify regulatory regions important for specific spatial domains. Four constructs gave mesodermal expression at very low frequency. The low frequency of mesodermal expression may result from the presence of incomplete regulatory elements.^ Shh is a signalling molecule which is normally restricted to the polarizing zone of the limb, interacts with the AER and is thought to play a major role in anterior-posterior patterning. The Shh expression pattern was altered by the use of the AER specific enhancer of the Msx-2 gene to direct ectopic expression of chicken Shh to the AERs of the limbs of transgenic mice. Ectopic expression of Shh results in the formation of limbs with deformities. Limb defects vary and include preaxial polydactyly, syndactyly, and oligodactyly. In the most severe cases the zeugopod and stylopod are also shortened and deformed. Hindlimb defects tend to be more severe than the forelimb defects, and preaxial hemimelia in the hindlimb is often observed. The expression patterns of several genes which are believed to be important in limb development and which interact with Shh were analyzed in the mutant limbs. Altered expression patterns for Msx-2, Fgf4, patched and Hoxd11 were observed in the mutant limbs.