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.

Fig 1. Expression of RBP7 mRNA in various chicken tissues.

A) Relative RBP7 mRNA expression values from chicken cDNA microarray (n = 3, pooled from three chickens for each tissue). B) Measurement of RBP7 mRNA expression in chicken tissues by qPCR (n = 4) which is normalized to β-actin as a housekeeping gene. S-Fat: subcutaneous fat, A-Fat: abdominal fat, T-Mus: thigh muscle, P-Mus: pectoralis major muscle.

Fig 2. Comparative analysis of RBP7 sequence and protein expression in avian species.

A) Comparison of RBP7 amino acid sequences of chicken (GenBank accession number XP_417606.4), turkey (XP_003212313.1), quail (corresponding to KP026122), human (NP443192.1) and mouse (NP071303.1). An epitope sequence detected by a custom RBP7 antibody is underlined from residue 73 to 83. B) Western blot analysis of RBP7 protein expression in chicken, quail and turkey tissues, respectively. α-tubulin was used as a loading control. SF: subcutaneous fat, AF: abdominal fat, TM: thigh muscle, PM: pectoralis major muscle, H: heart, Lu: lung, Li: liver, Ki: kidney, Sp: spleen, Br: brain, Int: intestine.

Fig 3. Analysis of RBP7 promoter, vector construct containing RBP7 promoter-eGFP, and PCR genotyping of transgenic quail.

A) RBP7 promoter with regulatory elements interacting with major transcription factors in adipose tissue. B) Schematic representation of the pLT-RBP7p3k-eGFP vector. Restriction sites, ClaI and PacI are depicted. LTR, long terminal repeat; ψ, packaging signal; RRE, rev-response element; cPPT, central polypurine tract of HIV-1; eGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. The arrows represent the location of forward and reverse primers used for genotyping PCR. C) Genotyping PCR of transgenic and wild-type quail using the two sets of primers depicted in the figure of B). WT, wild-type quail; +: pLT-RBP7p3k-eGFP plasmid DNA (positive control).

Fig 4. GFP expression in various tissues of transgenic quail.

A) GFP expression in selected quail lines under normal and UV light. 1. Neck fat; 2. Abdominal fat; 3. Muscle; 4. Heart; 5. Lung; 6. Liver; 7. Kidney; 8. Spleen; 9. Brain; 10. Intestine; 11. Abdominal skin; and 12. Wing skin. B) Comparison of GFP expression in neck fat and abdominal fat tissues.

Fig 5. Quantitative analysis of gene expression in stromal-vascular (SV) and fat cell (FC) fractions and GFP images of cultured primary cells from transgenic quail.

A) Quantification of RBP7, DLK1, FABP4, and PPARγ mRNA expression in SV and FC fractions from chicken adipose tissue by qPCR (n = 4). β-actin expression was used as a normalization control. B) qPCR analysis of SV and FC fractions from adipose tissue of selected transgenic G2 quail for mRNA expression of GFP, SCD1, FABP4, and RBP7 (n = 3). Ribosomal protein S13 (RPS13) was selected as a reference gene. C) Phase contrast and GFP images of primary preadipocytes and adipocytes from K5 and K7 transgenic G2 quail.