| 谢畅,于明举,惠楠,王岭斌,李文杰,赵永聚.2010.通过睾丸内注射转染外源DNA在小鼠精子的表达.动物学杂志,45(2):151-157. |
| 通过睾丸内注射转染外源DNA在小鼠精子的表达 |
| Testis Injection of Exogenous DNA Results in Expression in Mouse Sperm |
| 投稿时间：2009-09-10 修订日期：2009-12-28 |
| DOI： |
| 中文关键词: pEGFP 睾丸注射 精子介导基因转移 转基因小鼠 |
| 英文关键词:pEGFP Testis injection SMGT Transgenic mice |
| 基金项目:国家科技重大专项项目(No.2008ZX08008-004);国家自然科学基金项目(No.30600430) |
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| 中文摘要: |
| 为研究睾丸内注射外源DNA法生产转基因小鼠(Mus musculus)的可行性,并探讨注射DNA的最佳浓度。将环形的质粒DNA pEGFP-N1与脂质体混合制备DNA-脂质体复合物,按DNA浓度不同分为0.08μg/μl、0.12μg/μl和0.24μg/μl3组,分别注射入成年SPF级昆明小鼠睾丸内,同时设空白对照;每组处理公鼠2只,注射5d后每只与3只成年母鼠同笼,20d后在荧光显微镜下检测公鼠附睾精子,并制作睾丸石蜡切片,检测绿色荧光蛋白(GFP)的表达;PCR法检测各组后代阳性率。结果显示,3组小鼠附睾荧光精子比例分别为9.09%、47.06%和27.78%。3组小鼠的睾丸石蜡切片中均可看到不同强度的GFP表达。后代经PCR检测阳性率分别为17.26%、47.61%和22.11%。本实验证实了睾丸注射法能使外源DNA整合进入精子基因组,并能在自身和后代中得到表达,本研究中外源DNA注射浓度以0.12μg/μl效果为最佳。 |
| 英文摘要: |
| The aim of this study is to produce transgenic mice by testis-mediated gene transfer and investigate the suitable concentration of exogenous DNA for injection. A total of six SPF KM male mice were divided into three groups and liposome-treated exogenous DNA was directly injected into testis at three different concentrations:0. 08 μg /μl,0. 12 μg /μl and 0. 24 μg /μl,respectively. Every treated mouse was naturally mated with 3 female mice on the 5th day after the injection. The spermatozoa were detected on the 20th day after injection under the fluorescence microscope,and mouse testicular paraffin sections were prepared for detection of green fluorescent protein (GFP) expression under fluorescence microscope. The integration of pEGFP-N1 in the F1 was determined by PCR. The results showed that the proportion of florescent sperm in 3 groups were 9. 09% , 47. 06% and 27. 78% ,respectively. Expression of GFP in different degrees was also found in three groups of paraffin sections. The PCR positive rates of offspring were 17. 26% ,47. 61% and 22. 11% ,respectively. It is concluded that the transgenic mouse could be produced by direct injection of foreign DNA into mouse testis,and 0. 12 μg /μl was the suitable concentration of exogenous DNA for injection in this study. |
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