Chen Stephen, Choo Andre, Wang Nai-Dy, Too Heng-Phon, Oh Steve Kah Weng
Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, Singapore, Singapore.
Biotechnol Lett. 2007 Feb;29(2):261-5. doi: 10.1007/s10529-006-9223-3. Epub 2006 Nov 8.
FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.
用荧光素标记的寡聚脱氧胸苷酸（FAMdT）作为一种手段，来评估小核酸向小鼠胚胎干细胞（mESC）集落的有效转染。利用过夜生长的集落，转染主要局限于集落的周边，在同源的八聚体结合转录因子3/4（Oct-3/4）小干扰RNA（siRNA）递送后，Oct-3/4 RNA转录水平仅下降40%。然而，基于对约90%的Oct-3/4 RNA转录本敲低的定量实时PCR检测，接种后4小时对mESC进行转染，成功转染的细胞超过90%。该方法提供了一种经济有效的手段，用以确定有效的转染条件，并在据报道难以转染的细胞系（如mESC）中建立有效的siRNA敲低。
