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.

Figure 1

Photograph showing Plexiglas plastic inserts employed to align LV5 vertebral bodies (left) and femurs (right) inside sample holders for the Scanco µCT40 microCT scans. The red dots indicate placement of LV5s, with 12 rows of 4 columns. The lack femurs indicate placement of femurs, with three rows of six columns. Bones are fixed in place by adhering soft tissue and removable tape around the outside of the insert.

Figure 2

Venn diagram showing the numbers of KO lines undergoing HTS analyses for DEXA, LV5 microCT and femur shaft microCT.

Figure 3

Histograms showing distributions of vBMD (N=46 228 mice), LV5 Tb BV/TV (N=13 979 mice) and midshaft femur cortical thickness (N=13 845 mice) HTS values for all gene KOs examined, with s.d. indicated. Lines show normal distributions for the calculated means and s.d.s. HTS data for selected genes having clear bone phenotypes are indicted by arrows. s.d., standard deviation.

Figure 4

MicroCT scans of femurs from WT (left) and Sgpl1 KO (right) mice.

Figure 5

DKK1 antibody treatment in young mice. Data are means±s.e.m. for 10 mice per group treated between 4 and 8 weeks of age with antibody doses of 30 mg⋅kg⁻¹ weekly or a single 10 µg⋅kg⁻¹ zoledronate dose. Statistical evaluation involved ANOVA followed by Dunnett’s test for multiple comparisons.