Identification of the Avian RBP7 Gene as a New Adipose-Specific Gene and RBP7 Promoter-Driven GFP Expression in Adipose Tissue of Transgenic Quail

Abstract

The discovery of an increasing number of new adipose-specific genes has significantly contributed to our understanding of adipose tissue biology and the etiology of obesity and its related diseases. In the present study, comparison of gene expression profiles among various tissues was performed by analysis of chicken microarray data, leading to identification of RBP7 as a novel adipose-specific gene in chicken. Adipose-specific expression of RBP7 in the avian species was further confirmed at the protein and mRNA levels. Examination of the transcription factor binding sites within the chicken RBP7 promoter by Matinspector software revealed potential binding sites for adipogenic transcription factors. This led to the hypothesis that the RBP7 promoter can be utilized to overexpress a transgene in adipose tissue in order to further investigate the function of a transgene in adipose tissue. Several lines of transgenic quail containing a green fluorescent protein (GFP) gene under the control of the RBP7 promoter were generated using lentivirus-mediated gene transfer. The GFP expression in transgenic quail was specific to adipose tissue and increased after adipocyte differentiation. This expression pattern was consistent with endogenous RBP7 expression, suggesting the RBP7 promoter is sufficient to overexpress a gene of interest in adipose tissue at later developmental stages. These findings will lead to the establishment of a novel RBP7 promoter cassette which can be utilized for overexpressing genes of interest in adipose tissue in vivo to study the function of genes in adipose tissue development and lipid metabolism.

Adipose‐Specific Expression, Developmental and Nutritional Regulation of the Gene‐Encoding Retinol‐Binding Protein 7 in Pigs

“…To examine adipose‐specific expression of RBP7 in pigs as in other species including avians (Ahn et al, ) and mice (Ahn et al, ), RNA‐seq datasets were analyzed and Western blot analysis confirmed the expression level. Analysis of the RNA‐seq dataset (accession number GSE77776) revealed that the mRNA expression of RBP7 (Ensembl Gene ID: ENSSSCG00000003399) was approximately 70‐fold in porcine subcutaneous adipose tissue compared to seven other tissues including the heart, muscle, spleen, lung, liver, and kidney and highly statistically significant ( p < 0.001) (Fig.…”

“…A RBP7 polyclonal antibody was raised against an immunogen including amino acid 73–83 residues (EDNKGLDNRKC) of porcine RBP7 protein in the rabbit by a custom antibody service (AbClon, Seoul, Korea). Western blot analysis with tissue protein extracts was performed as described in our previous report (Ahn et al, ). In detail, equal amounts of protein extracts were loaded onto gels before wet‐transfer to PVDF membranes (Bio‐Rad, Hercules, CA, USA).…”

“…Previous studies have reported adipose‐specific genes that significantly influence adipocyte biology and possess etiological significance for obesity and obesity‐related diseases (Achari and Jain, ; Furuhashi et al, ; Hu et al, ; Hunt et al, ; Lefterova and Lazar, ; Li et al, ; Pan and Myers Jr., ; Tontonoz et al, ; Tsai and Maeda, ; Zhang et al, ). Our group has investigated genes abundantly expressed in adipose tissue that are related to lipid metabolism using the pig as a model (Ahn et al, ; Deiuliis et al, ; Li et al, ) as well as various animal species (Ahn et al, , ; Li et al, ; Song et al, ). Recently, adipose‐specific expression patterns and developmental regulation of the gene‐encoding retinol‐binding protein 7 (RBP7) were identified from avian and murine species by our group through comparative bioinformatics approaches and confirmatory analyses (Ahn et al, , ); however, its expression in adipose tissue of the porcine species has yet to be explored.…”

“…Our group has investigated genes abundantly expressed in adipose tissue that are related to lipid metabolism using the pig as a model (Ahn et al, ; Deiuliis et al, ; Li et al, ) as well as various animal species (Ahn et al, , ; Li et al, ; Song et al, ). Recently, adipose‐specific expression patterns and developmental regulation of the gene‐encoding retinol‐binding protein 7 (RBP7) were identified from avian and murine species by our group through comparative bioinformatics approaches and confirmatory analyses (Ahn et al, , ); however, its expression in adipose tissue of the porcine species has yet to be explored.…”

“…Because retinol is an essential nutrient that possesses diverse functions in animal growth, reproduction, and vision (Kanai et al, ), the role of RBP involved in retinol homeostasis is important for animal biology and physiology. Among RBP, RBP7 was identified as a protein abundantly produced by adipocytes across species (Ahn et al, , ; Tsutsumi et al, ). As de novo lipogenesis mainly occurs in adipose tissue in the case of pigs (Gondret et al, ; Mildner and Clarke, ) and adipose tissue is one of the major reservoirs for retinol and its metabolites (Flajollet et al, ; Frey and Vogel, ), analyses of expression patterns of RBP7 in adipose tissue as a RBP is essential to understand adipose development and lipid metabolism in pigs.…”