Fraga D, Yano J, Reed M W, Chuang R, Bell W, Van Houten J L, Hinrichsen R
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington.
J Eukaryot Microbiol. 1998 Nov-Dec;45(6):582-8. doi: 10.1111/j.1550-7408.1998.tb04553.x.
A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelia has been developed. For these studies antisense oligonucleotides directed to different regions of the calmodulin mRNA were used. It was found that a pulse delivered at 150-250 V (375-625 V/cm field strength) for 3.9-4.2 ms using a 275 microF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 microM. In addition, multiple oligodeoxynucleotides directed to the same target mRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity.
已经开发出一种利用电穿孔将反义寡脱氧核苷酸导入四膜虫的方法。在这些研究中，使用了针对钙调蛋白mRNA不同区域的反义寡核苷酸。结果发现，使用275微法的电容器，电阻设置为13欧姆，在150 - 250伏（场强375 - 625伏/厘米）下施加3.9 - 4.2毫秒的脉冲，足以使高达95%的处理细胞中实现荧光标记的寡脱氧核苷酸的可测量掺入。最佳参数包括使用长度至少为12个碱基且带有3' 封闭基团的寡脱氧核苷酸，剂量约为10微摩尔。此外，针对同一靶mRNA的多个寡脱氧核苷酸可使实现预期效果所需的寡脱氧核苷酸剂量至少降低10倍。综上所述，这些结果表明，使用反义寡脱氧核苷酸可能是一种将基因与四膜虫特定功能联系起来的简便而有用的方法。最后，本报告讨论了不同的3' 封闭基团以及针对同一靶mRNA不同区域的寡脱氧核苷酸组合的使用如何有助于解决特异性问题。
