精子介导转基因动物的制备

采用直接注射的方法，将脂质体包裹的含人乳铁蛋白基因的重组质粒pLNCXHLF注入兔睾丸组织和羊输精管中，一个月后分别与正常雌兔及正常的母羊交配。所产仔兔均为活体，经PCR和Southern检测,转基因仔兔阳性率平均为35%(11/31)；羔羊经引物F1/R1系统PCR检测阳性率为33.3％（4/12），F2/R2系统PCR检测阳性率为25%（3/12）。将流产羔羊组织解剖后提取舌、肺、肝脏、肌肉、皮肤、脑、生殖腺、脾脏、小肠、心脏、肾脏共11种组织的总DNA进行PCR检测，发现：F1/R1和F2/R2系统PCR检测出阳性信号存在于不同组织；F2/R2 PCR系统检测阳性信号较F1/R1 PCR系统少；而且各种组织的外源DNA存在的拷贝数不等，造成阳性信号的强弱程度不同。结果表明：1）脂质体包裹外源基因转染精子的方法，可将外源基因导入受精卵，并得到了较高的转基因阳性率；2）精子携带外源DNA的整合过程是随机的，在受精过程和胚胎早期分化过程中可能发生了片段丢失、不完全整合或游离于基因组存在而产生嵌合体。本文的研究结果证明了该方法是一种简捷有效的新途径，为进一步深入探讨精子介导转基因动物制备可行性奠定了基础，给精子载体法制备转基因哺乳动物研究提供了有价值的参考。 Abstract:The recombinant plasmids pLNCXHLF fused with human lactoferritin gene were directly injected into male rabbit's testis and male goat's spermaductus.The transfected males were fertilized with females one month later.Using F1/R1 PCR system and southern blotting,the transgenic positive rate of rabbit offsprings genomic DNA was 35%(11/31).From the PCR results of F1/R1 and F2/R2 system,the transgenic positive rate of genomic DNA of goat offsprings were 33.3% (4/12)and 25%(3/12) respectively.We prepared genomic DNA from 11 kinds of tissues of goat offsprings,which were tongue,lung,liver,muscle,skin,brain,gonad,spleen,intestines,heart,and kidney.The result of F1/R1 PCR system indicated that the abilities to uptake exogenous DNA were various in different tissues;the positive signals of F2/R2 PCR system were feebler than the ones of F1/R1 PCR system,and the density of positive signals attributed to the amount of copies of exogenous DNA in the tissues.In this experiment,spermatozoa-mediated gene transfer can produce the transgenic animals after exogenous DNA being entrapped by liposome.But during the course of fertilization and the early process of embryo proliferation,the exogenous DNA had lost segments,partly integrated,or existed outside of genomic DNA.So the rate of chimera was relatively high.According to this result,this method is not only a simple and effective way to produce transgenic animals but also make references to other researchers to prepare transgenic mammals by this means.

Research on Spermatozoa-mediated Gene Transfer to Product Transgenic Animals

The recombinant plasmids pLNCXHLF fused with human lactoferritin gene were directly injected into male rabbit's testis and male goat's spermaductus.The transfected males were fertilized with females one month later. Using F 1/R 1 PCR system and southern blotting,the transgenic positive rate of rabbit offsprings genomic DNA was 35%(11/31).From the PCR results of F 1/R 1 and F 2/R 2 system,the transgenic positive rate of genomic DNA of goat offsprings were 33.3% (4/12)and 25%(3/12) respectively.We prepared genomic DNA from 11 kinds of tissues of goat offsprings,which were tongue,lung,liver,muscle,skin,brain,gonad,spleen,intestines,heart,and kidney.The result of F 1/R 1 PCR system indicated that the abilities to uptake exogenous DNA were various in different tissues;the positive signals of F 2/R 2 PCR system were feebler than the ones of F 1/R 1 PCR system,and the density of positive signals attributed to the amount of copies of exogenous DNA in the tissues.In this experiment,spermatozoa mediated gene transfer can produce the transgenic animals after exogenous DNA being entrapped by liposome.But during the course of fertilization and the early process of embryo proliferation,the exogenous DNA had lost segments,partly integrated,or existed outside of genomic DNA.So the rate of chimera was relatively high.According to this result,this method is not only a simple and effective way to produce transgenic animals but also make references to other researchers to prepare transgenic mammals by this means.