Establishment?Identification And Application Of Rabbit Permanent Melanocytes

The coat color of mammals mainly depends on the deposition of melanin,and the production of melanin is mainly regulated by melanocytes.Melanocytes produce different types of melanin,and the difierent distribution of these pigments leads to the formation of a variety of coat color types in mammals.In the modem industry of fiir rabbits,the white coat needs to be dyed before it is made into clothing and other products,which will affect the environment and the health of corsumers.However,the pure natural color coats have no this problem,and they are more favored by consumers.To solve this problem,it is necessary to deeply anatyze the pigmentation mechanism of rabbit coat,and provide a theoretical basis for transformation of coat color and breeding of new coat color.However,the immortalized melanocytes lines of rabbits have not yet been established,which leads to the lag in the research on the formation mechanism of rabbit for color and the mechanism of melanin deposition This study is focused on the establishment and identification of immortalized cell Knes of rabbit melanocytes,and which is used as a material to preliminarily analysis the key genes of pigment deposition-small eye malformation associated transaction fector's(microphthalmia-associated transcription factor gene)patterns of expression and function.The immortalized melanin cell lines provide a cell model in vitro for studying the melanin depositional law and mechanism of rabbit fur,and lay the foundation for further revealing the molecular mechanism of pigment deposition in for animals.The main research results are as follows:1.According to the establishment of the in vitro culture system of rabbit melanocytes in the earfy stagp of the research groiy,the primaiy rabbit skin melanocytes were separated by two-step enzyZe digestion method,and on basis of which,more pure melanocytes were obtained after purification and screening.pLVX-RES-Puro-SV40LT lentivirus expression vector was constructed,and the infection efficiency was the highest when Multiplicity of Infection was 100 according to the lentivirus packaging and identification.Lentivirus was used to infect primary melanocytes for 72 h,and was screened using 2.0 ?g/mL puroraycin for 1 week,then was maititained using 1.0 ?g/mL puromycin for 2 weeks,and a monoclonal cell was selected,1 strain of rabbit melanin cell line with more than 50 passage of culture algebra was obtained after expanded culture,.2.The morphology of immortalizsed cells maintained tte basic morphological characteristics of primary cells by microscopic observation,showing a multipolar shape with strong refraction;The cells were stained with L-DOPA,and showed brown and black granules.The immunofluorescence staining of S-100,TYR,TYRP1 and MITF were all positive,indicating that the established immortalized cells still had the characteristics of nornal melanocytes.It was showed that the proliferation activity of melanin cell lines was consistent with that of primary cells,and the proliferation level remained normal,according to the growth curve and cell cycle of melanocytes lines;Karyotype analysis assay was performed,and showed that the number of ehromosomes in the immortalized cells was 44,which was consistent with the nurber of normal primary melanocytes and still had normal karyotype;In the soft agar experiment,the melanocytes lines in suspension did not undergo division and proliferation,and tumor nasses were not found in the nude mice injected,which was no different from normal melanocytes cells,indicating that the immortalized cells did not have canceration and could be used as tool cells.3.According to NCBI prediction and reports of the literature,the specific primers of MITF coding region were designed,and six variable splices of MITF gene were identified by the methods ofRT-PCR and TA clonal sequencing,which were named as MITF XI,MITF X2,MITF X3,MITF X4,MITF X5 and MITF X6(Genbank accession numbers:MK 801776,MK 825547,MK 85548,MK 825549,MK 825550 and MK 825551).The over-expression vectors of different spliceosomes of MITF were constructed and transfected into the immortalized melanocytes,and according to RT-qPCR assay,it was showed that the over-expression of MITF XI-X3 significantly increased the mRNA expression levels of TYR and GPNMB(P<0.05),whae the over-expression of other MITF spliceosomes had no significant efect on the downstream genes(P>0.05).The determination of melanin content showed that MITF XI-X6 significantly increased the deposition amount of total melanin and eumelanin of melanocytes(P<0.05),indicating that these spliceosomes could affect the melanin deposition of melanocytes.In conclusion,the established immortalized melanocytes of rabbit can be used as an ideal in vitro cell model to replace the primary cells for the study of pigmentation in fur animals.