RESUMO
Sperm-mediated gene transfer (SMGT) is one of the most methods in the transgenic animal research and the efficiency of spermatozoa binding and internalization exogenous DNA after sperm/DNA co-culture is important to a successful SMGT.In this study,the influencing factors of exogenous DNA uptake by spermatozoa were detected using DIG labeled EGFP as exogenous gene.The results demonstrated that porcine spermatozoa could spontaneously take up exogenous DNA which mainly binding occurs on the sub-acrosomal and nuclei region of the sperm head.The rate of spermatozoa binding exogenous DNA increased with the extending action of time.At 37℃ and 39℃,the rate of spermatozoa uptake exogenous DNA would not increase after 60 min incubation,and the similar result was observed on 90 min at 17℃.Binding rates and internalization rates of washed ejaculated sperm cells from the 15 boars varied between 6.57%-35.81% and 2.990%-24.66%,respectively.The binding rate and intemalization rate were mostly inhibited by seminal plasma.The binding rates were significantly increased by liposome and DMSO,respectively.Dead-spermatozoa could bind exogenous DNA,the intermalization process could not be completed.Furthermore,the highest binding rate was found in membrane broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors.