Recent advances have brought the power of modern molecular genetic techniques to the Paramecium researcher. This, coupled with its utility for a classical genetics approach, provides the researcher with a variety of useful tools for dissecting the molecular details of a physiological process.
For a long time, one of the great advantages of Paramecium was the ease with which one could generate mutants of a given phenotype. The power of this approach has been enhanced by the recent description by Kung and colleagues of a protocol for cloning by complementation in Paramecium (Haynes et al., 1998). This approach combines microinjection, DNA separation, and cloning techniques to fish out and identify a complementing gene from genomic DNA. It has already been successfully used to identify the genes responsible for previously described mutant phenotypes.
After identifying a gene responsible for a function, it is often advantageous to introduce the gene or an altered version back into an organism to better understand its function. The process of introducing DNA into a cell and having it expressed is referred to as transforming the cell. Transformation is possible in Paramecium using either of two different approaches. The first approach is to directly inject the DNA into the macronucleus using finely pulled micropipette needles. The DNA is injected into the macronucleus where it is amplified and stably maintained through several fission cycles (Godiska et al., 1987). If a suitable promoter is attached to the gene, it can be expressed. Two recent developments now allow researchers to introduce DNA into cells using bioballistics or electroporation (Boileau et al., 1999). Bioballistics involves literally shooting DNA into the cell using high velocity particles such as gold or tungsten onto which the DNA has been coated by precipitation. By chance, some of the particles wind up in the macronucleus and the DNA is released from the particles, amplified and then expressed. Electroporation is similar but uses a high voltage electric field to open temporary holes in the cell membrane through which DNA or other particles can traverse. In either case, to identify the few cells that have taken up transforming DNA and expressed the encoded genes requires a powerful selection technique. In Paramecium DNA is typically introduced that contains an antibiotic resistance gene (usually paromomycin resistance). Thus transformants are identified because only they can survive incubation with the antibiotic.
It is also desirable to knock out or silence specific genes in an organism as a way to understand what the gene does physiologically. This is also possible in Paramecium using a variety of techniques. The two easiest methodologies are the use of antisense oligonucleotides and RNA interference (Fraga et al., 1998; Ruiz et al., 1998). In both cases the target gene sequence needs to be known. Each method essentially knocks out or silences a specific gene allowing investigators to ask what effect the silencing has on the physiological process under investigation. Both methods are relatively easy to use and promise to keep Paramecium an important model system for some time to come.
Boileau, A. J., Kissmehl, R., Kanabrocki. J. A., and Saimi, Y. Transformation of Paramecium tetraurelia by electroporation or particle bombardment. J. Euk. Microbiol. 46:56-65.
Fraga D., Yano, J., Reed, M. W., Chuang, R., Bell, W., Van Houten, J. L. and Hinrichsen, R. 1998 Introducing antisense oligonucleotides into Paramecium via electroporation. J. Euk. Microbiol. 45:582-588.
Godiska r., Aufderheide, K. J., Gilley, D., Hendrie, P., Fitzwater, T., Preer, L. B., Polisky, B., and Preer J. R. Jr. 1987 Transformation of Paramecium by microinjection of a cloned serotype gene. Proc. Natl. Acad. Sci USA 94:1310-1315.
Haynes W.J., Ling K.Y., Saimi Y., Kung C., 1996. Toward cloning genes by complementation in Paramecium, J. Neurogenet. 11:81–98.
Hinrichsen R. D., Fraga, D. and Reed, M. W. 1995 3’ modified antisense oligonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium. Proc. Natl. Acad. Sci. USA 89:8601-8605.
Ruiz F., Vayssie L., Klotz C., Sperling L., Madeddu L., 1999 Homology-dependent
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