Figure 3. Jumpstarter method in Harmonia axyridis.

(A) Crossing scheme for jumpstarter method. A mutator line and a jumpstarter line are crossed (G₁). Newly hatched larvae expressing both ECFP and DsRed transformation markers in the CNS were selected from the progeny (G₂). Larvae carrying both the mutator and jumpstarter elements were heat shocked to induce transposase expression. Newly hatched larvae expressing only the DsRed transformation marker were selected from the progeny (G₃), and analyzed by PCR for mobilization of the mutator (i.e. integration elsewhere in the genome). Each unique insertion line was used to establish a new mutator line. (B) Schematic representation of PCR method to analyze mobilization of the original mutator element. Primer set HaG1 and PLR was used to detect the original non-mobilized mutator element. If the original mutator element is remobilized, no PCR product will be detected. Primer set HaG1-HaG3 was used as a positive control to detect the homologous, mutator element-free chromosome. (C) Red circles denote progeny with apparent remobilization events. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle, because the original mutator element was mobilized to other genomic sites. These larvae were established as new mutator lines. Each G₂ female gave at least one newly mobilized progeny. (D) Comparison of DsRed fluorescence between original mutator line (middle) and two new mutator lines (left and right). Compare to original mutator line (middle), two new mutator line larvae show higher (left) or lower (right) expression of DsRed. (E) PCR analysis of mutator element mobilization. Newly hatched G₃ larvae strongly or weakly expressing DsRed marker compared with original mutator line were selected and subjected to PCR analysis. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle. In this case, detection efficiency of remobilized mutator element is increased compared with random selection (see result in C). (F) An enhancer-trap line of transgenic ladybird beetles. During the generation of mobilized mutator lines using the jumpstarter method, an enhancer expressing GFP throughout the body was detected (left); a wild-type control larva is also shown on the right. Panel displays a ventral view (anterior uppermost).