CAJ | 학술논문

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60 min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.
정자개도기인전이(SMGT)시목전전기인동물연구중간단이고효적방법지일,기중정자결합화내화외원기인적효솔시정자개도전기인성공적관건.본시험이DIG표기적선성화EGFP작위시종기인래검측저정자결합화내화외원기인적영향인소,결과표명:저정자능구자발결합외원기인,결합부위주요재정자정체후구.정자결합외원DNA적양성솔수공부육시간연장이증가,재37℃혹39℃시부육60 min후,양성솔불재증가;재17℃시부육90min후,양성솔불재증가.재검사적15지공저양본중,정자여외원DNA적결합솔위6.57%～35.81%,내화솔위2.99%～24.66%,개체간차이현저(P<0.01).정장능구강렬억제외원기인적결합화내화,지질체급DMSO능구현저제고전염효솔.사정자능구결합외원기인,단불능장기내화;반복동융도치질막파렬적정자대외원기인유경고적결합솔,차불수개체영향.
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.