1. Introduction
Alpacas are a member of the camelid family from South America, where they live in the semi-arid areas of the high-altitude plateau. They are famous for their soft fleece, which is known for its good strength and a range of natural coat colors from solid white to black. Not only does the coat color of the alpaca have important economic value, alpaca is also a perfect animal model in which to study the mechanisms controlling mammalian coat color genes.
Mammalian coat colors are formed by melanocytes produce melanin in skin, hair bulbs and eyes[1]. The pigmentation function of the melanocyte is highly conserved in mammalian evolution. Lin et al. (2007) have reported melanocytes are located in the epidermis and the dermis[2]. Melanocytes …show more content…
Protein concentrations in cell lysates were determined spectrophotometrically using the NanoDrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Heat-denatured protein samples (300ng per lane) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Boster, Wuhan, China). The membranes were incubated for 60 min in 10% nonfat dry milk to block nonspecific binding, followed by incubation for 12 h at 4℃with primary antibody [primary mouse monoclonal antibody against MITF(Thermo, CA, USA), primary rabbit monoclonal antibody against TYR (Abcam, England), primary rabbit monoclonal antibody against TYR1 (Abcam, England), primary rabbit monoclonal antibody against TYR2 (Abcam, England)], which was diluted 1:2,000 in tris-buffered saline Tween-20 (TBST), or with a mouse monoclonal antibody against β-actin (Tansgen Biotech, Beijing, China) diluted 1: 3,000 in TBST. The membrane was then washed 2 times for 10 min in TBST, 1 time for 10 min in TBS, and incubated for 1 h at room temperature with a goat anti-rabbit second antibody or a goat anti-mouse second antibody conjugated to horseradish peroxidase (Cwbio, Beijing, China) diluted 1:1700 in TBST. The membrane was washed 2 times for 10 min in TBST and 1 time for 10 min in TBS, and the bound antibody was detected colorimetrically using a superstar ECL plus …show more content…
More than 100 pigmentation genes affected melanocyte development, in which MITF was a conserved member of the Myc-related family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors. It had been implicated as a master gene for the development and survival of melanocytes. Additionally, MITF was thought to be a key transcription factor for the regulation of the expression of major melanogenic proteins such as tyrosinase and tyrosinase-related proteins[6, 14-16]. The 3’ UTR of MITF gene had over 3 kb in the mouse, and 35% of the bases were conserved in the mouse 3’UTR compared with all the species. So long conserved sequence in 3’UTR suggested that the region might have a regulation function[17]. At the 3’UTR of MITF, many miRNAs binding sites were found by publicly available algorithms (TargetScan 5.2, www.targetscan. org; miRBase, microrna.sanger.ac.uk), but these miRNAs exact functions targeted MITF were not very clearly. Tian et al. (2012) had reported miR-148a-3p expression in the alpaca skin, and higher expression in white coat color than in brown coat color with the aid of Illumina sequencing technology, the fold change of miRNAs between white and brown alpaca skin was 2.58 [12]. In 293T cell, we found miR-148a-3p could interact with the 3’UTR MITF though luciferase assay, according to the data, we concluded the binding sites of miR-148a-3p was mainly binding site 2. Though interacting
Alpacas are a member of the camelid family from South America, where they live in the semi-arid areas of the high-altitude plateau. They are famous for their soft fleece, which is known for its good strength and a range of natural coat colors from solid white to black. Not only does the coat color of the alpaca have important economic value, alpaca is also a perfect animal model in which to study the mechanisms controlling mammalian coat color genes.
Mammalian coat colors are formed by melanocytes produce melanin in skin, hair bulbs and eyes[1]. The pigmentation function of the melanocyte is highly conserved in mammalian evolution. Lin et al. (2007) have reported melanocytes are located in the epidermis and the dermis[2]. Melanocytes …show more content…
Protein concentrations in cell lysates were determined spectrophotometrically using the NanoDrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Heat-denatured protein samples (300ng per lane) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Boster, Wuhan, China). The membranes were incubated for 60 min in 10% nonfat dry milk to block nonspecific binding, followed by incubation for 12 h at 4℃with primary antibody [primary mouse monoclonal antibody against MITF(Thermo, CA, USA), primary rabbit monoclonal antibody against TYR (Abcam, England), primary rabbit monoclonal antibody against TYR1 (Abcam, England), primary rabbit monoclonal antibody against TYR2 (Abcam, England)], which was diluted 1:2,000 in tris-buffered saline Tween-20 (TBST), or with a mouse monoclonal antibody against β-actin (Tansgen Biotech, Beijing, China) diluted 1: 3,000 in TBST. The membrane was then washed 2 times for 10 min in TBST, 1 time for 10 min in TBS, and incubated for 1 h at room temperature with a goat anti-rabbit second antibody or a goat anti-mouse second antibody conjugated to horseradish peroxidase (Cwbio, Beijing, China) diluted 1:1700 in TBST. The membrane was washed 2 times for 10 min in TBST and 1 time for 10 min in TBS, and the bound antibody was detected colorimetrically using a superstar ECL plus …show more content…
More than 100 pigmentation genes affected melanocyte development, in which MITF was a conserved member of the Myc-related family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors. It had been implicated as a master gene for the development and survival of melanocytes. Additionally, MITF was thought to be a key transcription factor for the regulation of the expression of major melanogenic proteins such as tyrosinase and tyrosinase-related proteins[6, 14-16]. The 3’ UTR of MITF gene had over 3 kb in the mouse, and 35% of the bases were conserved in the mouse 3’UTR compared with all the species. So long conserved sequence in 3’UTR suggested that the region might have a regulation function[17]. At the 3’UTR of MITF, many miRNAs binding sites were found by publicly available algorithms (TargetScan 5.2, www.targetscan. org; miRBase, microrna.sanger.ac.uk), but these miRNAs exact functions targeted MITF were not very clearly. Tian et al. (2012) had reported miR-148a-3p expression in the alpaca skin, and higher expression in white coat color than in brown coat color with the aid of Illumina sequencing technology, the fold change of miRNAs between white and brown alpaca skin was 2.58 [12]. In 293T cell, we found miR-148a-3p could interact with the 3’UTR MITF though luciferase assay, according to the data, we concluded the binding sites of miR-148a-3p was mainly binding site 2. Though interacting