Notebook

Summary

Establishment of engineering Drosophila is a time-consuming task. The following is a record of the process of our project experiment. For detailed operation procedures and experimental results, please refer to the Protocols and Engineering pages

1 January

(weekends & winter vacation):

Constructions of vectors of pUAST-MTF-1, pMRE-Hid and pMRE-GFP

  1. Primer design and synthesis
  2. Total RNA extraction
  3. Reverse transcription
  4. PCR and product recovery
  5. Digestion and purification
  6. Overnight ligation
  7. Transformation
  8. Positive colony identification
  9. Sequencing

2 February – March

(winter vacation & weekends):

1.Microinjection of Drosophila embroy

  • Plasmid extraction
  • Sending plasmid samples for Drosophila embryo injection.

2.Cell biologyexperiments

2.1 Co-transfection of plasmids into Drosophila S2 cells

  • Recovery of S2 cells
  • Co-transfection of plasmids into cells using transient transfection reagent
  • Culturing of cells in media with different concentrations of metal ions

2.2 Real-time PCR

  • Collecting cells and extracting total RNA
  • Reverse transcription
  • Real-time PCR

2.3 Western blot detection of MTF-1 and Hid genes transcription and expression

  • Collecting and lysing cells after transfection for 48 hours
  • Preparation of SDS-PAGE gel
  • SDS-PAGE electrophoresis
  • Membrane transfer
  • Blocking
  • Hybridization (overnight)
  • Development

3 April - June

(weekends) :

1.Establishment of stable genetic strains of Drosophila

2.Pre-experiment of Drosophila hybridization

  • Drosophila medium preparation
  • Common culture of Drosophila
  • Dissection and observation of Drosophila larvae
  • Dissection and observation of adult Drosophila
  • Cultivation of Drosophila in medium with different concentrations of metal ions

4 July

(summer vacation):

3.Drosophila hybridization

  • Hybridizing Drosophila strains GMR-GAL4/ Vg-GAL4/ptc-GAL4 and UAS-MTF-1；MRE-Hid or UAS-MTF-1；MRE-GFP
  • Culture hybrid oosperm in media with different concentrations of metal ions

4.Phenotypic observations of Drosophila larvae

  • Larva dissection and identification of imaginal disks
  • AO staining
    1. Staining
    2. make slide and photography
    3. c.Counting of bright spots
  • Dcp-1 staining
    1. Fixation of larval tissue
    2. Cleaning of larval tissue
    3. Low-temperature incubation with primary antibody and cleaning
    4. Staining with secondary antibody solution and cleaning
    5. Dissection and make slide
    6. Observation and photography under fluorescence microscope.
    7. Counting of bright spots
  • GFP observation
  • Literature review

5 August

(summer vacation) :

Phenotypic observations of Drosophila adults

  • Culture hybrid oosperm in media with different concentrations of metal ions
  • Collect adult flies of F1 generation
  • Observe the eye and wing phenotypes and take photos
  • Calculate eye size and wing size of flies from each metal ion concentration treatment