Introducing antisense oligodeoxynucleotides into Paramecium via electroporation

A method utilizing electroporation to deliver antisense oligodeoxynucleotid es into Paramecium tetraurelia has been developed. For these studies antise nse oligonucleotides directed to different regions of the calmodulin mRNA w ere used. It was found that a pulse delivered at 150-250 V ( 375-625 V/cm f ield strength) for 3.9-4.2 ms using a 275 mu F capacitor with resistance se t at 13 Ohms was sufficient to achieve measurable incorporation of fluoresc ently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Opti mal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 mu M. 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 metho d for linking genes to specific functions in Paramecium tetraurelia. Finall y, this report discusses how different 3' blocking groups and the use of co mbinations of oligodeoxynucleotides directed to different regions of the sa me target mRNA can help address concerns about specificity.