Introducing antisense oligodeoxynucleotides into Paramecium via electroporation.
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.
Fraga
D
D
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington. dfraga@acs.wooster.edu
Yano
J
J
Reed
M W
MW
Chuang
R
R
Bell
W
W
Van Houten
J L
JL
Hinrichsen
R
R
D000818:Animals; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D020319:Oligodeoxyribonucleotides, Antisense; D016805:Paramecium tetraurelia / Q000235:genetics*
D002147:Calmodulin; D020319:Oligodeoxyribonucleotides, Antisense