Transformation of Nematodes by Microinjection

Abstract

The free-living nematode, Caenorhabditis elegans, has been an important model system in the study of developmental and cell biology. Significant advances in mapping and sequencing the C. elegans genome have aided our understanding of fundamental biological processes. Development of an efficient method for gene transfer has been a key tool accelerating C. elegans research advances (Kimble et al., 1982; Stinchcomb et al., 1985., Fire, 1986; Mello et al, 1991). Integrative transformation in C. elegans is reproducibly achieved after microinjecting DNA directly into maturing oocyte nuclei (Fire, 1986). Heritable extrachromosomal DNA transformation in C. elegans was first described by Stinchcomb et al. (1985) after microinjecting DNA into the gonad cytoplasm. DNA molecules injected into the cytoplasm of the C. elegans hermaphrodite gonad undergo a transient period of reactivity. This results in the formation of large heritable extrachromosomal structures that experience very little further rearrangement (Mello et al., 1991). Germ cell nuclei in C. elegans develop initially in a syncytium (a multinucleate mass of protoplasm resulting from fusion of cells) and when cell membranes later envelop them, the exogenously added DNA is packaged into the oocyte. Transforming DNA is generally not integrated into the chromosomes, but rather is maintained as a concatamer (i.e. unite in a chain) of introduced sequences.