High‐throughput Screening of Mouse Knockout Lines Identifies True Lean and Obese Phenotypes

Abstract

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were ≥1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

Mechanistic principles of RAF kinase signaling

“…The calcineurin interaction allows KSR2 to activate ERK signaling following an increase in intracellular calcium [40]. KSR2 regulates cellular energy metabolism in part through its interaction with AMPK, and this contributes to the obese phenotype of KSR2 knockout mice [39,41].…”

LX4211 Increases Serum Glucagon-Like Peptide 1 and Peptide YY Levels by Reducing Sodium/Glucose Cotransporter 1 (SGLT1)–Mediated Absorption of Intestinal Glucose

“…The Institutional Animal Care and Use Committee at Lexicon Pharmaceuticals approved all study protocols. General methods for mouse husbandry (Brommage et al, 2008), generation of SGLT12/2 mice (Powell et al, 2013), and generation of SGLT22/2 mice (Jurczak et al, 2011) have been described. Unless stated otherwise, all mice studied were of mixed genetic background (129/ SvEvBrd and C57BL/6-Tyrc-Brd); genotyping was performed on tail DNA as described previously (Donoviel et al, 2001).…”

Sustainable Three-Dimensional Tissue Model of Human Adipose Tissue

“…3 Currently, adipose tissue biology is mainly studied in the context of cell culture monolayers in in vitro culture, or with small animal in vivo studies. [4][5][6][7][8] Both methods offer insight to adipose systems, yet neither approximates the complex nature of human adipose tissue.…”