Team:KIT-Kyoto/Notebook-week5p

September 20th

1-1-64 Transfection

At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP DNA was added to the well. As a negative control, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-EGFP DNA alone was added to the well. Similarly, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-LacZ DNA was added to the well. As a negative control for this experiment, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-LacZ DNA alone was added to the other well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 or 72 hours at 25 ℃.

September 22nd

The cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i).
Results: 12.6% cells were observed to be EGFP-positive, while only o.5% cells were apparently EGFP-positive for the negative control cells, indicating that the P-element plasmid, pUAS-EGFP DNA, Biobrick part (BBa_K758006) is truly functional in Drosophila cells. We therefore decided to send this prasmid to iGEM Headquarters.

Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA
Right panel: cells transfected with pUAS-EGFP DNA alone (negative control)

　　　　　　

The cells transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were fixed with cold methanol for 20 min or 4% paraformaldehyde for 15 min. After fixation, cells were washed with 0.5% Triton X-100 in PBS, then masked with 5% normal goat serum in PBS for 1 hour. Then the monoclonal anti-beta galactosidase antibody (X500 dilution in PBS containing 5% normal goat serum) was added to the fixed cells and incubated for 16 hours at 4℃.

September 23rd

After incubation with the first antibody, the cells were washed with PBS (three times for 10 min each). Then the Alexa-conjugated anti-mouse IgG (X400 dilution in PBS containing 5% normal goat serum) added to the cells and incubate for 4 hours at 4℃. After washing with PBS (three times for 10 min each), cells were mounted with mounting medium in DAPI and examined under the confocal lazer microscope (Olympus, Fv10i). Results: 10.1% cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were observed to be LacZ-positive, while no LacZ-positive cell was detected for the negative control cells, indicating that the P-element plasmid, pUAS-LacZ DNA, Biobrick part (BBa_K758005) is truly functional in Drosophila cells. We therefore decided to send this plasmid to iGEM Headquarters.

After 72 hours incubation, the cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i). Results were nearly the same as those after 48 hours incubation.

Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA
Right panel: cells transfected with pUAS-LacZ DNA (negative control)