Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2010) 21 OC4.4

SFEBES2009 Oral Communications Bone and parathyroid (8 abstracts)

Rapid screening for novel bone phenotypes in 100 consecutive lines from the Wellcome Trust Sanger Institute Gene Targeting Programme

Apostolos Gogakos 1 , Duncan Bassett 1 , Anne van der Spek 1 , Holly Evans 2 , Jacqui White 3 , Ramiro Ramirez-Solis 3 , Karen Steel 3 , Allan Bradley 3 , Rajesh Thakker 4 , Peter Croucher 2 & Graham Williams 1

1Molecular Endocrinology Group, Imperial College London, Hammersmith Hospital, London, UK; 2Mellanby Centre for Bone Research, University of Sheffield, Sheffield, UK; 3Mouse Gene Targeting Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK; 4Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, UK.

The Wellcome Trust Sanger Institute Gene Targeting Programme is deleting all mouse genes and has already generated 400 knockout mice in a C57/BL6N background with a further 4000 genes targeted in ES cells. Two hundred and fifty new knockouts will undergo limited phenotyping each year. However, the programme lacks a sensitive and sufficiently detailed screen for individual physiological systems, each of which requires high throughput methodology and unique expertise. Thus, we prospectively investigated the sensitivity and reproducibility of two screening methods for identification of abnormal skeletal phenotypes in 100 consecutive knockout strains. Upper and lower limbs and caudal vertebrae from 15-week-old mutant females (n=2–6 per strain) were compared to genetic background controls (n=94). Mutants with known skeletal defects were covertly included as positive controls. Faxitron MX20 images (10 micron resolution) of limbs and vertebrae were calibrated to three internal standards and bone lengths, cortical thickness and mineral content were determined. Trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and bone mineral density (BMD) in proximal tibia were determined by microCT (Skyscan 1172a, 4.3 micron resolution). Thus far, 55 lines have been analysed by Faxitron and 76 by microCT. Faxitron identified at least one abnormality in 23% of strains, including abnormalities of bone length (4%) and femur (13%) and vertebra (17%) mineral content. Micro-CT identified at least one abnormal trabecular bone parameter in 22%, including abnormalities of BV/TV (14%), Tb.Th (7%), Tb.N (14%) and BMD (6%). In 54 strains analyzed by both methods concordance was 78%. Undisclosed strains with known skeletal abnormalities were identified correctly. Abnormalities in discordant strains were limited to parameters determined by only one technique, thus demonstrating complementarity between the two methods. This pilot study predicts that 20% of knockout mice will have skeletal abnormalities that can be identified and evaluated sensitively and specifically by high throughput screening.

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