Genetic Transformation of a Mouse Cell Line with Genes of the Receptor for Equine Infectious Anemia Virus (ELR1) and Cyclin T1 (eCT1)
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The complexity of the pathogenesis and insufficient knowledge of the equine infectious anemia virus (EIAV) make it necessary to find an adequate laboratory model for studying the infectious process and the immune response. One of the ways to solve this problem is the production of cell cultures with the desired properties using genetic transformation. The aim of the present study was to obtain mouse embryonic fibroblasts with genes of the receptor of the equine infectious anemia virus (ELR1) and cyclin T1 (eCT1). For this purpose, cells were transfected with pcDNA3.1-ELR1 and pcDNA3.1-cycT1 plasmids containing nucleotide sequences of the target genes using the calcium-phosphate method. Eight stable genetically transformed clones of STO cells with [ELR1eCT1neo]+ phenotype were obtained using the selection of cells on G418. The frequency of genetic STO transformation was 2.8 × 10–5. The integration of genes into the cell genome was detected by PCR using gene-specific primers with subsequent nucleotide sequencing using DNA isolated from cells as the matrix. The expression of equine ELR1 and eCT1 genes in mouse fibroblasts was confirmed at the transcriptional level by the presence of the mRNA of the target genes in the RT-PCR reaction. The obtained stable STO cell line was deposited in the cell culture collection of the All-Russia Scientific Research Institute of Experimental Veterinary Medicine Federal Scientific Center.
Keywordsequine infectious anemia virus gene of the receptor for equine infectious anemia virus equine cyclin gene transfection genetic cell transformation mouse embryonic fibroblasts in vitro
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