All animal experiments were approved by the Institutional Animal Care and Use Committee of Amgen (animal protocol #2006-00010). Mice were housed in a pathogen-free facility with a 12 h light-dark cycle. Mice were allowed ad libitum access to water and food. They were fed with normal chow (Harlan 2920) before starting a high fat diet (Research Diets D12451, 45 kcal % fat).

Gpr21 KO mice were created using a pair of transcription activator-like effector nucleases (TALENs) from Life Technologies targeting exon 2 of mouse Gpr21 TALEN binding sites are underlined below with a 15 base pair spacer between the 2 sites.

5 ’- TGAACTCCACCTGGGATGG TAATCAGAGCAGCCA TCCTTTCTGTCTTCTGGCA

ACTTGAGGTGGACCCTACC ATTAGTCTCGTCGGT AGGAAAGACAGAAGACCGT - 5’

Design, cloning and validation of the TALENs were performed by Life Technologies. Messenger RNA (provided from Life Technologies) for each of the TALENs were diluted in RNAse free microinjection buffer to a final concentration of 4.0 ng/µl for each TALEN (8.0 ng/µl total concentration). The TALENs were microinjected into the pronucleus of fertilized one-cell embryos (0.5 days post coitus) obtained from the mating of C57BL/6 (Taconic) males to superovulated C57BL/6 (Taconic) female mice. Microinjected eggs were transferred to pseudopregnant Swiss Webster recipients. Founder pups were screened for TALEN induced mutations in Gpr21 by sequencing across exon 2. Two founders, one with a 5 bp deletion and the other with a 29 bp deletion were expanded for further analysis.

Genomic DNA was prepared from liver, BAT and spleen using DNeasy blood and tissue kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. PCR was carried out using the primers 5’-CAGCATGAAGTGAGAGCCAG-3’ and 5’-CAAGTAGCCCAGTGCCAGAAG-3’.

For the microarray analysis, mRNA was isolated from 6 animals for each group using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) and processed following the protocols described in section 2 (Eukaryotic Sample and Array Processing; 701024 rev 1) of the Affymetrix Technical Manual. Briefly, 5 µg total RNA was used to synthesize cDNA (10 pmol of T7-(dT)24 primer, and Superscript II (Invitrogen, Carlsbad, CA). Purified double-stranded cDNA (MinElute Reaction Cleanup Kit, Qiagen, Valencia, CA) was used to generate biotinylated cRNA using Bioarray HighYield RNA Transcript labeling Kit (Enzo Diagnostics, Farmingdale, NY) followed by purification with Qiagen RNeasy Mini kit and hybridization to the Affymetrix HT MG 430 PM array. Arrays were washed on a GeneChip Fluidic Station 450 (EukGE_WS2v4_450 protocol) and scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). Data analysis was conducted with R (version 2.15, http://r-project.org), Bioconductor (version 2.10, http://bioconductor.org/) and ArrayStudio (Omicsoft, version 8.0). Briefly, Affymetrix CEL files were normalized in Bioconductor using the GCRMA method. Differentially regulated genes were identified using a moderated t-test. False discovery rate adjusted P-values were calculated using the method of Benjamini & Hochberg (1995).

Total RNA was isolated using Qiagen midi RNA preparation kit (Qiagen, Valencia, CA). The total RNA concentration was determined with a Nanodrop (ThermoFisher Scientific, Wilmington, DE). QPCR was performed using 10 ng RNA per well, Taqman master mix (Applied Biosystems, Foster City, CA). Rabgap1 gene expression was assessed using Taqman probes from Applied Biosystems (Mm01327207_m1 and Mm01327199_m1). Gpr21 mRNA level was measured using the forward primer 5’-CACCTGGGATGGTAATCAGAG-3’, reverse primer 5’- TCACAATGATGTTGCCAGAAAT-3’ and probe 5’ FAM/TTCTGGCAC/Zen/TGGGCTACTTGGAAA/IABkFQ-3’ from Integrated DNA Technologies (Coralville, IA). Results were evaluated using the ΔΔC _T method and normalized relative to the expression of glyceraldehyde-3-phosphate dehydrogenase using QuantStudio 6 and 7 Flex Real-Time PCR System Software in a QuantStudio™ 7 Flex System.

At 6 am, mice were fasted for 4 hr. Glucose levels were measured and blood samples were taken from the tail vein before oral glucose tolerance test (OGTT) was initiated. GTT was performed by oral administration of a bolus glucose (2g/kg body weight). Glucose levels were measured at 20, 40 and 60 min after glucose administration by using AlphaTrak blood glucose meter (Abbott, Chicago, IL).

Blood samples were collected from tail vein when mice were at 11 weeks old, 15 weeks old (on HFD for 4 weeks) and 26 weeks old (on HFD for 15 weeks) Terminal blood was collected by cardiac puncture. Blood samples were centrifuged at 10000 rpm. Serum insulin levels were determined by using Insulin (mouse) ultra-sensitive EIA kit 80-INSMSU-E10 or mouse high range insulin ELISA 80-INSMSH-E01 (ALPCO Diagnostics, Salem, NH).

We isolated RNA from spleen, liver, perirenal fat (WAT) and brown fat (BAT) from Deltagen Gpr21 KO mice and their wildtype littermate control mice. Microarray results identified that Rabgap1 was the only gene that was changed in all the tissues analyzed. Rabgap1 mRNA levels were increased by 1–4 fold when using two independent Rabgap1 probes ( Figures 1A, B, Table 1) located upstream of Gpr21 gene and were down regulated by 5–15 fold when using one Rabgap1 probe located downstream of Gpr21 gene. This result indicated that the genetic modifications in the original KO line modified Rabgap1 mRNA expression levels ( Figure 1B). Strbp mRNA expression levels were not changed when using multiple probes that were located upstream and downstream of the Strbp gene ( Figure 1A).

A new line of Gpr21 KO mice ( Gpr21 TAL 29bp) was created by deleting a 29 bp within the coding exon of Gpr21 gene. Using the TALENS technology, a 29 bp very close to the ATG codon was deleted, thus causing an out of frame mutation and early termination of Gpr21 gene ( Figure 2A). Homozygous Gpr21 KO mice genotype was confirmed by PCR using primers located upstream and downstream of the 29bp deletion from genomic DNA of several tissues ( Figure 2B). As predicted, a 100 bp band was identified for the wildtype mice and a 71 bp band for the Gpr21 TAL 29 bp homozygous KO mice ( Figure 2C). The PCR fragment was sequenced and a 29 bp deletion was confirmed. From RNA isolated out of BAT and liver, no detectable Gpr21 mRNA levels were identified in Gpr21 TAL 29 bp KO mice using qPCR and Gpr21 probes that are located around the 29 bp deletion region ( Figure 2D). Similar mRNA levels were detected using primer/probe that were located downstream of 29 bp deletion in exon 2 (data not shown). The result confirmed that Gpr21 transcripts in Gpr21 TAL 29bp KO mice had the 29 bp deletion.

Rabgap1 mRNA expression levels were assessed using 2 Taqman probes ( Table 1) that amplified different regions of Rabgap1 in liver and BAT of KO and their wildtype littermate mice. One primer/probe set spanned Rabgap1 exon 3 and 4 ( Table 1), which is located upstream of the Gpr21 gene. Another primer/probe set spanned Rabgap1 exon 17 and 18, which is located downstream of the Gpr21 gene ( Table 1). Rabgap1 mRNA expression levels in Gpr21 TAL 29 bp KO mice were not changed compared with their wildtype littermate mice with both primer/probe sets ( Figure 3A). Liver and BAT Strbp mRNA expression levels were also not changed between Gpr21 TAL 29bp KO mice and their wildtype littermate mice ( Figure 3B).

The body weight, OGTT and insulin levels of Gpr21 TAL 29 bp KO mice fed a normal chow were not different from the ones of their wildtype littermates ( Figures 4A–C). Mice were then fed with a 45% high fat diet to induce obesity and insulin resistance. After 4 weeks and 15 weeks of high-fat feeding, Gpr21 TAL 29 bp KO mice gained similar body weight to that of their wildtype littermates, showed no difference on glucose tolerance and fasting blood glucose and insulin levels were not different from their wildtype littermates ( Figures 4D–I), respectively.

The results of Osborn and Gardner suggest that GPR21 may play an important role in regulating body weight and glucose metabolism. However, in our attempts presented here to replicate their findings we didn’t see the same effect. We would therefore like to encourage an open discussion in the wider community to further elucidate the potential effectiveness of pharmacologically inhibiting GPR21.