Abstract: This study was aimed to establish a system of buffalo primary somatic lentivirus infection.The efficiency and fluorescence intensity of buffalo granulosa cell (BuGC),buffalo mammary epithelial cell (BuMEC) and buffalo fibroblast cell (BuFC) infected by lentivirus were compared.The optimal multiplicity of infection (MOI) values of three buffalo primary somatic cells were selected.BuGC,BuMEC and BuFC were isolated and cultured.HEK293T cell was transfected with pLVX-Puro-GFP,pSPAX2 and pMAD2.G by liposome,and then the virus was collected at 48 and 72 h after transfection,and the titer of lentivirus was determined by dilution counting method.BuGC,BuMEC and BuFC were exposed to different multiplicity of infection (100,200,400,600 and 800) of the lentivirus.After 72 h,the cells were observed by the inverted fluorescence microscope,the transfection efficiency and fluorescence intensity were calculated.Three buffalo primary somatic cells were infected with lentivirus and screened with puromycin.The results showed that at MOI ≤ 200,the fluorescence intensity of cells after lentivirus infection of BuMEC was the strongest,and there was no significant difference between BuGC and BuFC;At MOI ≥ 400,the fluorescence intensity of cells after lentivirus infection was BuMEC>BuGC>BuFC.At MOI=100,the lentivirus infection efficiency was BuGC>BuMEC>BuFC;At MOI=200,the lentivirus infection efficiency of BuMEC and BuGC were 100%.At MOI=800,the lentivirus infection efficiency of BuFC was 100%.Different cell types had different tolerance to lentivirus.Under the selection and maintenance of puromycin,three buffalo primary somatic cell lines with overexpression of green fluorescent protein (GFP) in buffalo primary somatic cells were obtained.The optimal MOI of BuMEC,BuGC and BuFC infected by lentivirus were 200,400 and 800,respectively.All three buffalo primary somatic cells were able to establish the cell lines which overexpressed stably a foreign gene with lentivirus.This results provided good cell models for the study of buffalo breast tissue development and differentiation,lactation regulation,reproductive development and preparation of transgenic animals.

Key words: buffalo; lentivirus; granulosa cell; mammary epithelial cell; fibroblast