[Frontiers in Bioscience E3, 158-165, January 1, 2011]

Repression of Slc24a5 can reduce pigmentation in chicken

Xiao-fang Liu, Juan Luo, Xiao-xiang Hu, Hui Yang, Xiao-qing Lv, Chun-gang Feng, Jia Tong, Yan-qiang Wang, Shao-hua Wang, Xiao-juan Liu, Tong-hui Lin, Jing Fei, Ying Liu, Ning Li

State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100193, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods 3.1. Chicken embryonic RPE cell preparation
3.2. siRNA synthesis, transfection and psiCHECK-2-Slc24a5 construction
3.3. Quantitative RT- PCR
3.4. Western blot
3.5. Measurement of melanin production
3.6. Electron microscopic analysis using photoelectron emission microscopy 4.
Results 4.1. Identification of siRNA that cause knockdown of Slc24a5 mRNA
4.2. siRNA-5 as an effective suppressor of Slc24a5 expression
4.3. Melanin production reduced by down-regulation of Slc24a5 using RNAi in chicken RPE cells
4.4. Knock-down of the Slc24a5 gene reduced TYR
4.5. Melanosomes were affected by a Slc24a5 knock-down in chicken RPE cells
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Slc24a5 is a putative cation transporter, which is a member of the potassium-dependent sodium-calcium ion exchanger family. Association of the Slc24a5 gene with pigmentation has been established in Zebrafish, mice and humans. Despite these findings, its function in chicken remains unknown. The intent of this study was to describe the association of Slc24a5 with respect to melanin deposition in the chicken using RNAi. The objective was to detect the variety of melanin deposition caused by the down-regulation of Slc24a5 in chicken retinal pigment epithelium (RPE) cells. Nine siRNAs that targeted against Slc24a5 mRNA were found to be effective in suppressing Slc24a5 gene expression in 293FT cells. The most effective target tested effectively inhibited Slc24a5 expression in mRNA and subsequently reduced protein levels in RPE cells. These results show that down-regulation of Slc24a5 results in a reduction of melanin content, as well as a decrease of melaeneous type Ⅲ and type Ⅳ melanosomes simultaneously. Taken together, this work suggests that Slc24a5 function is conserved in the chicken, and necessary for melanin synthesis.