High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Abstract

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets.

Adult Tph2 knockout mice without brain serotonin have moderately elevated spine trabecular bone but moderately low cortical bone thickness

“…11 Previous studies 11,13 examined Tph2 KO mice at 4 to 16 weeks of age, whereas Lexicon's mice were examined at 18 through 83 weeks of age. Tph2 KO mice have a transient growth retardation starting a few days after birth and lasting through 4 months of age 18,19 and limited data from Lexicon's KO mouse screening analyses 16 support this finding. Low trabecular bone Modest skeletal phenotypes in adult Tph2 KO mice R Brommage et al mass in studies examining growing mice is possibly related to their small size.…”

“…Data obtained during highthroughput screening. 16 Equal numbers of male and female mice were examined for each genotype at each age, with Ns ¼ 4 for WT and Ns ¼ 8 for KO mice. Po0.001 at 2 weeks and P ¼ 0.02 at 4 weeks of age.…”

“…16 Given continued interest in serotonin and bone, 17 we examined bone mass in additional cohorts of adult Tph1 KO, Tph2 KO and Tph1/Tph2 DKO mice.…”

MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization

“…These transporters control the uptake and efflux of many critical ions or substrates during amelogenesis and have been determined to result in enamel abnormalities if mutated. Mutations in SLC4A4 result in pigmentation differences and a propensity to fracture (21,(42)(43)(44)(45)(46). Patients with proximal renal tubular acidosis (pRTA) caused by mutations in SLC4A4 also exhibit dental abnormalities (5,42).…”

Constitutively elevated blood serotonin is associated with bone loss and type 2 diabetes in rats

“…However, an association among LRP5 deficiency, circulating 5HT and bone loss has not been reproduced in mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans [3]. Further, association between circulating serotonin and bone mass has not been unequivocally confirmed in different mouse knockout models (global knockouts of TPH1 and TPH2, LRP5) [4][5][6]. De Vernejoul and colleagues revisited the bone phenotype in mice with genetic deletion of peripheral 5HT-synthesizing enzyme tryptophan hydroxylase-1 (TPH1 -/-) and showed that osteoclasts synthesize 5HT which acts to induce osteoclast precursor differentiation in a local micro-serotoninergic system via a mechanism of RANKL-induced osteoclast formation [7].…”