小鼠睾丸注射不同转染试剂和注射方法对外源基因表达的影响

Effect of different transfection reagents and injection methods in mice testicular injection on the expression of exogenous gene

摘要:

为探讨不同转染试剂 (LipofectamineTM LTX & PLUSTM、Lipofectamine2000和纳米化聚酰胺-胺型树枝状聚合物 (PAMAM-D)) 和睾丸注射方法 (睾丸网注射、曲精细管注射和间质注射) 对转基因小鼠生产效率的影响，将pEGFP-C1质粒分别与不同转染试剂混合后，按照不同的注射方法注入小鼠睾丸内，30 d后检测小鼠精子密度、活力、精子阳性率以及配种后仔鼠转基因阳性率。结果3种转染试剂对小鼠繁殖性能影响由小到大依次为LipofectamineTM LTX & PLUSTM、Lipofectamine 2000和PAMAM-D。转染后LipofectamineTM LTX & PLUSTM、Lipofectamine 2000和PAMAM-D组精子的GFP阳性率分别为35.65%±0.69%、12.86%±0.35%和10.04%±0.20%。配种后仔鼠的PCR阳性率分别为29.17%、13.70%和5.88%。三种不同注射方法对小鼠睾丸都造成损伤由小到大依次为睾丸网注射、曲精细管注射和睾丸间质注射，3者的阳性精子比例分别为35.13%±1.727%、15.13%±1.457%和0%，配种后仔鼠的PCR阳性率分别为33.3%、12.5%和0%。结果表明，LipofectamineTM LTX & PLUSTM和睾丸网注射对小鼠睾丸的损伤最小，并能获得较高的转染效率。

Abstract:

The purpose of this study was to study the effect of three different transfection reagents (LipofectamineTM LTX & PLUSTM, Lipofectamine 2000 and Nano-PAMAM-D) and three different testicular injection methods (rete testicular injection, seminiferous tubules injection and testicular interstitial injection) on the efficiency of production transgenic mice. After the mixtures of plasmid DNA (pEFP-C1) and transfection reagent were injected with different testicular injection methods, the sperm density, vitality, positive sperm rates and PCR positive transgenic mice rate were examined 30 days after injection. The results showed that the damage degree from slight to serious of three transfection reagents was LipofectamineTM LTX & PLUSTM, Lipofectamine 2000, and PAMAM-D. The sperm positive rates with green fluorescence of these three groups were 35.65%±0.69%, 12.86%±0.35% and 10.04%±0.20%, respectively. The PCR positive rates of transgenic newborn mice were 29.17%, 13.70% and 5.88%, respectively. Among the groups of different testicular injection methods, the damage degree from slight to serious was rete testicular injection, seminiferous tubules injection, and testicular interstitial injection, whereas the sperm positive rates with green fluorescence were 35.13 %, 15.13%, and 0%, respectively. The PCR positive rates of transgenic newborn mice among different testicular injection groups were 33.3%, 12.5%, and 0.0%. The combination of rete testicular injection and LipofectamineTM LTX & PLUSTM had the lowest toxicity and highest transgenic efficiency in the production of transgenic mice.