Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Zhao Zhao,Bao Bao,Liu Liu,Zhou Zhou,Hu Hu,Wu Wu,Li Li,Wang Wang,Yang Yang,Chen Chen

doi:10.3390/ijms20194874

Abstract

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Highlights

Epidermal melanocytes (MCs) are specialized melanin-producing cells that synthesize melanin within the melanosome [1,2,3,4]
The primary melanocytes of rabbits were isolated by two-step enzymatic digestion
The lentiviruses were infected into HEK293 cells and gene expression was confirmed by RT-qPCR

Summary

Introduction

Epidermal melanocytes (MCs) are specialized melanin-producing cells that synthesize melanin within the melanosome [1,2,3,4]. The coat color in mammals is controlled by melanin distribution and type [5,6]. White coats must be dyed prior to the production of clothing and other products, which impacts the environment and consumer health. Natural colored coats are most popular with consumers. The analysis of rabbit coat pigmentation provides a theoretical basis for the breeding of new coat color varieties. To-date, immortalized rabbit melanocytes (Im RMCs) have not been established, limiting our knowledge of melanin deposition in rabbits

Methods

Results

Conclusion