ABSTRACT
Three reasons can be advanced for the emergence of a successful non-drosophilid transformation technology over the past 4 years . One has been the availability of cloned marker genes and conesponding mutations in the target species . and the more recent use of the green fluorescent protein as a dominant selectable marker gene in insect species in which there is a paucity or absence of genetic mutations (Chalfie et al., 1994; Zwiebel et al., 1995; Cornel et al., 1997) . The second has been the recognition and acceptance that the P transposable element of Drosophila nzelcrmgasfer cannot be used to transform other insect species . This directly led to a search Eor other class I1 transposable elements (i.e., elements that transpose via a DNA intermediate) that might be useful gene vectors in insects . The discovery of the Mirzos (Frana and Savaltis. 1991). Hemzes (Atkinson
BGUWE 12.1 Interplasmid transposition assays in insect embro) os. For interplasmid transposition assays, three plaslnids consisting of a donor plasmid, a target plasmid. and a helper plasmid are microinjected into preblastoderm insect embryos. During embryonic development, the assay measures the ability of the transposase enzyme encoded by the helper plasmid to mediate the transpositon of the transposable element located on the donor plasmid to the target plasmid. The result is the piasmid shown in uhish only transposable element sequences have inserred. by transpositional recombination. into the target plasmid. Plasmids are recovered from developed emblyos -1 day after injection and transfoimed by electroporation into appropriate strains of E coli that are then plated on defined media. Plasmid DNA is prepared from colonies displaying the desired phenotype and then analyzed for the presence of the element in the target plasmid. The breakpoints of transposition are determined by DNA sequencing. Key: Kmr, Cam'. Ampf each refer to E. coli genes conferring resisiance to kanamycin. chloramphenicoi, and ampicillin: lacZ refers to the gene encoding 0-galactosidase: ori refers to the E. coli ColEl origin of replication. In the example shown, the target plasmid is delived from and cannot replicate in E coli. Arrows above and below the transposed element repreient sequencing primer sites.
