Transgene-associated human growth hormone expression in pancreatic (cid:2) -cells impairs identiﬁcation of sex-based gene expression differences

Trans-gene-associated human growth hormone expression in pancreatic (cid:2) -cells impairs identiﬁcation of sex-based gene expression differences. Am J Physiol Endocrinol Metab 316: E196–E209, 2019. Fluorescent protein reporter genes are widely used to identify and sort murine pancreatic (cid:2) -cells. In this study, we compared use of the MIP-GFP transgene, which exhibits aberrant expression of human growth hormone (hGH), with a newly derived Ins2 Apple allele that lacks hGH expression on the expression of sex-speciﬁc genes. (cid:2) -Cells from MIP-GFP transgenic mice exhibit changes in the expression of 7,733 genes, or greater than half of their transcriptome, compared with (cid:2) -cells from Ins2 Apple/ (cid:3) mice. To determine how these differences might affect a typical differential gene expression study, we analyzed the effect of sex on gene expression using both reporter lines. Six hundred ﬁfty-seven differentially expressed genes were identiﬁed between male and female (cid:2) -cells containing the Ins2 Apple allele. Female (cid:2) -cells exhibit higher expression of Xist , Tmed9 , Arpc3 , Eml2 , and several islet-enriched transcription factors, including Nkx2-2 and Hnf4a , whereas male (cid:2) -cells exhibited a generally higher expression of genes involved in cell cycle regulation. In marked contrast, the same male vs. female comparison of (cid:2) -cells containing the MIP-GFP transgene revealed only 115 differentially expressed genes, and com- parison of the 2 lists of differentially expressed genes revealed only 17 that were common to both analyses. These results indicate that 1 ) male and female (cid:2) -cells differ in their expression of key transcription factors and cell cycle regulators and 2 ) the MIP-GFP transgene may attenuate sex-speciﬁc differences that distinguish male and female (cid:2) -cells, thereby impairing the identiﬁcation of sex-speciﬁc variations. wk of age. Islets were lysed in the TETG buffer (20 mM Tris·HCl, pH 8.0, 1% Triton X-100, 10% glycerol, 140 mM NaCl, 2 mM EGTA) with 1 (cid:7) protease inhibitors (P8340; Sigma) by repeated freezing and thawing cycles. Final lysates were cleared by centrifugation and used for hGH radioimmunoassay (07151102; MP Biomedicals) performed in triplicates by Vanderbilt Hormone Assay and Analytical Services Core. Results were normalized to total protein content. Islet isolation and perifusion. Islet isolation and perifusion exper-iments were performed by the Vanderbilt Islet Procurement and Analysis Core. Islets were isolated by injection of 0.6 mg/ml Colla-genase P (Roche) into the pancreatic bile duct, and partially dissoci-ated tissue was fractionated using a Histopaque-1077 (Sigma) gradi-ent followed by hand-picking of islets. For FACS and RNA-Seq, islets from 3 to 7 mice were pooled for each sample. Glucose-stimulated insulin secretion was measured using islets from 4 wild-type male C57BL/6J mice (acquired directly from Jackson) and 4 Ins2 male mice bred at Vanderbilt (99.6% C57BL/6J) that were both 12 wk of age. A parallel, 4-column apparatus, maintained at 37°C by water immersion, was used to perifuse 50 size-matched islets from 4 different mice simultaneously. Islets were perifused with (5.6 glucose perifusion media (DMEM without glucose, pH 7.4, 38.1 sodium bicarbonate, 4 mM L -glutamine, 1 sodium pyruvate, 0.5% phenol red, 5.0 HEPES, and 0.1% BSA) 30-min equilibration period (baseline) before fractions for min in low (5.6 mM) glucose, high ferentially expressed genes generated from RNA-Seq data (9). False discovery rate-adjusted P value of 0.05 was used as a cutoff to declare signiﬁcance. Statistical analysis. Statistical signiﬁcance was determined using two-tailed Student’s t -test. A threshold of P 0.05 declare signiﬁcance.