Sato Fuminori, Soh Tomoki, Hattori Masa-Aki, Fujihara Noboru
Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka 8128581, Japan.
Asian J Androl. 2003 Sep;5(3):213-6.
To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents.
A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light.
The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents.
The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.
为了证实使用射出的鸡精子生成转基因鸡过程中外源基因的稳定性，对射出精液的精浆中的脱氧核糖核酸酶（DNase）活性进行了评估，并通过添加脂质转染试剂检测了DNA的稳定性。
使用pEGFPN-1载体的PCR片段（249 bp）作为DNA底物，在40℃下与精浆孵育30分钟。然后，将整个反应溶液进行琼脂糖凝胶电泳，并在紫外光下评估DNA大小。
与精浆孵育后，DNA底物完全消失。然而，与热处理的精浆孵育或在0.5 mmol/L至5 mmol/L EDTA存在下与精浆孵育后，底物保持完整。通过添加市售脂质转染试剂，底物在精浆中得以稳定。
射出的鸡精液的精浆中存在DNase活性。然而，DNA在脂质体-DNA复合物中是稳定的。
