Our team aims to develop a convenient and inexpensive visual monitoring system for heavy metals by constructing transgenic Drosophila. We inserted MTF-1 gene driven by UAS and Hid gene driven by MRE in Drosophila S2 cells, respectively. These two transgenic Drosophila lines interbreed and the offspring with both MTF-1 and Hid genes cross with the Drosophila line with genotype GMR-GAL4 or Vg-GAL4 or ptc-GAL4 which express GAL4 during eye/wing development. In this way, we constructed three Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4 which express MTF-1 during eye/wing development due to the GAL4-UAS system. In response to heavy metals in the environment, MTF-1 is activated, binds to MRE and activates Hid expression, leading to apoptosis in the developing eyes/wings. Thus, in the first generation within 5-10 days, these lines show abnormally small eye/wing sizes observable by naked eyes.
Our team successfully constructed 9 parts. BBa_K4890001, BBa_K4890004, BBa_K4890011, and BBa_K4890013 were developed for expressing MTF-1 gene driven by UAS. BBa_K4890002- BBa_K4890004, BBa_K4890012 and BBa_K4890014 were developed for expressing Hid gene driven by MRE. Plasmids based on these parts and pAc-GAL4 plasmid were co-transfected into Drosophila S2 cells to verified the efficiency of this monitor system in response to heavy metals. The cells was assessed by PCR, gene sequencing, real-time PCR and Western blot. Engineering based on these plasmids and hybridization were used to construct Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4. Treated with heavy metal ions, eye/wing imaginal discs from larvae were evaluated by AO staining and Dcp-1 staining and their adults’ eye/wing areas were analyzed. BBa_K4890015 was developed for expressing GFP gene driven by MRE. Engineering based on this part and hybridization were used to construct Drosophila line with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4. Treated with heavy metal ions, fluorescence intensity in the eye imaginal discs from larvae was analyzed.
| No. | Part Number | Type | Description | Length |
| 1 | BBa_K4890001 | Basic | MTF-1 | 2535 |
| 2 | BBa_K4890002 | Basic | MRE | 73 |
| 3 | BBa_K4890003 | Basic | Hid | 1537 |
| 4 | BBa_K4890004 | Basic | Hsp70 | 262 |
| 5 | BBa_K4890011 | Composite | UAS-Hsp70 | 342 |
| 6 | BBa_K4890012 | Composite | MRE-Hsp70 | 320 |
| 7 | BBa_K4890013 | Composite | UAS-Hsp70-MTF1 | 2724 |
| 8 | BBa_K4890014 | Composite | MRE-Hsp70-Hid | 1556 |
| 9 | BBa_K4890015 | Composite | MRE-Hsp70-GFP | 1032 |