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A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelin has been developed. For these studies antisense oligonucleotides directed to different regions of the calrnodulin 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 pF 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 )LM. In addition, multiple oligodeoxynucleotides directed to the same target rnRNA 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. Supplementary key words. Calcium-dependent K' channel, calcium-dependent Na' channel, calmodulin, cholesterol-linked oligo- deoxynucleotides, hexanol-linked oligodeoxynucleotides, protozoan behavior. NTISENSE oligodeoxynucleotides (ODN) can be used to A transiently down-regulate a gene's expression and thereby help identify the role of the gene product in the cell (2). Their ease of use makes antisense ODN an attractive method for uti- lizing a "reverse genetics" approach for the assignment of function to cloned genes. Previously, we described the micro- injection of antisense ODN as a viable methodology in the cil- iated protozoan, Paramecium tetraurelia (5). In that report, an- tisense ODN were targeted against calmodulin mRNA resulting in abbreviated behavioral responses that mimicked the type of behavioral responses seen in cells with a mutant calmodulin gene that does not properly activate the calcium-dependent Na+ current. Thus, it was concluded that the antisense ODN reduced the levels of calmodulin sufficient to affect the activation of the calcium-dependent Na+ current. Recently it was reported that electroporation can be used as a method for incorporating ODN into cells (l) and, in an effort to improve the ease of use of this technology in ciliates, we have developed a procedure for introducing antisense ODN by electroporation. Calmodulin was again chosen as the target be- cause of its well-characterized role in the control of behavior in Paramecium (6, 141. In addition, we have characterized some of the parameters important for the use of this methodology in Paramecium. |
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