Ouatas T, Le Mével S, Demeneix B A, de Luze A
Laboratoire de Physiologie Générale et Comparée, Muséum National d'Histoire Naturelle, URA 90 CNRS, Paris, France.
Int J Dev Biol. 1998 Nov;42(8):1159-64.
The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.
聚乙烯亚胺（PEI）阳离子聚合物与质粒DNA的配方已得到优化，可在体内将基因导入非洲爪蟾蝌蚪脑。通过脑室内微量注射1微升（含0.5至1微克DNA），我们发现线性低分子量聚合物22 kDa PEI比支链25 kDa聚合物的效率显著更高。带有轻微正净电荷的复合物（由每个DNA磷酸基团与6个PEI胺形成的比例）提供了最佳的转染水平。转基因表达呈DNA剂量依赖性，并在6天内保持，即实验的时间进程。使用β-半乳糖苷酶构建体检查空间分布，发现表达该转基因的神经元遍布整个大脑。通过检查三碘甲状腺原氨酸（T3）对哺乳动物TRH和Krox-24启动子序列转录的影响，探讨了使用该技术评估生理调节的可能性。向水族箱水中添加生理浓度的T3可显著降低大鼠TRH启动子的转录，而相同处理则增加了小鼠Krox-24 -荧光素酶构建体的转录。因此，PEI-DNA转染为在蝌蚪脑转录水平上追踪生理调节提供了一种通用且易于应用的方法。
