猪精子结合和内化外源基因的影响因素研究  

Influencing factors of porcine spermatozoa binding and internalization exogenous DNA

作　　者：杜欢[1] 杨继山[1] 翟向玮[1] 孙理兰[1] 柳丽华[1] 沈伟[1] 闵令江[1] 潘庆杰[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109

出　　处：《中国兽医学报》2009年第7期933-938,共6页Chinese Journal of Veterinary Science

基　　金：山东省教育厅项目(J05K06);青岛市科技计划资助项目(06-2-2-11-jch);青岛农业大学高层次人才启动基金重点资助项目(630515630615)

摘　　要：精子介导基因转移（SMGT）是目前转基因动物研究中简单而高效的方法之一，其中精子结合和内化外源基因的效率是精子介导转基因成功的关键。本试验以DIG标记的线性化EGFP作为示踪基因来检测猪精子结合和内化外源基因的影响因素，结果表明：猪精子能够自发结合外源基因，结合部位主要在精子顶体后区。精子结合外源DNA的阳性率随共孵育时间延长而增加，在37℃或39℃时孵育60min后，阳性率不再增加；在17℃时孵育90min后，阳性率不再增加。在检查的15只公猪样本中，精子与外源DNA的结合率为6．57％～35．81％，内化率为2．99%～24．66％，个体间差异显著（P〈0．01）。精浆能够强烈抑制外源基因的结合和内化，脂质体及DMSO能够显著提高转染效率。死精子能够结合外源基因，但不能将其内化；反复冻融导致质膜破裂的精子对外源基因有更高的结合率，且不受个体影响。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 coulture 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 rain incubation,and the similar re- sult 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. 99%- 24. 66%, respectively. The binding rate and internalization rate were mostly inhibited by seminal plasma. The binding rates were significantly increased by liposome and DM- SO, 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.

关 键 词：精子介导基因转移 猪 精子 外源基因 

分 类 号：S828[农业科学—畜牧学]