Figure 5. (A) Schematic representation of the piggyBac donor and helper plasmids constructed using Manduca sexta Polyubiquitin gene promoter to drive the expression of DsRed and EGFP marker and the piggyBac transposases. (B) Manduca sexta embryos showing transient DsRed and EGFP expression at 48-75 h after microinjection.
To work on piggyBac approach, first we isolated Manduca sexta Polyubiquitin (MsexPUb) gene promoter, which is widely used in other insects for transgenesis. To check the MsexPUb promoter activity in the Manduca embryos, we have constructed two piggyBac donor plasmid to drive the expression of DsRed and EGFP marker under the control of MsexPUb gene promoter; MsexPUb-EGFP and MsexPUb-DsRed (Fig. 5A). Then these plasmids were injected individually in 1-hr old Manduca embryo to check the transient expression. We confirmed the transient expression of MsexPUb-EGFP and MsexPUb-DsRed in 48 and 72 h old Manduca embryos and in the first instar larvae under the fluorescence microscope using GFP and RFP filter (Fig. 5B). After confirming the MsexPUb promoter activity, next we have made insect-codon optimized piggyBac transposes helper plasmids using the MsexPUb promoter 1) MsexPUb-iPBase and 2) MsexPUb-ihyPBase. Then, piggyBac donor and helper plasmid mixture were injected into the Manduca embryos for germline transformation. So far, we have not recovered the transgenic Manduca sexta. In the future, we will work on optimizing this system to develop a germline transformation protocol for Manduca sexta.