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Team:KIT-Kyoto/Project
From 2012.igem.org
Project Overview
We try to develop new medicine for therapy of leukemia which is more free from side effects.
For this study, we are going to use Drosophila melanogaster, a model organism to establish transgenic fly carrying responsible genes for human leukemia.
Introduction
Drosophila melanogaster has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.
Results
BBa_K758006(UAS-EGFP)
We transfected this plasmid cultured cells of Drosophila under different two conditions. We also transfected pGaTB plasmids which express GAL4 proteins one cultured cells and we didn’t the other cells.
If our BBa_K758006 was assembled accurately, this plasmid expresses enough EGFP under activation of GAL4 protein. So cultured cells that were also transfected pGaTB have fluorescence strongly, and ones transfected no pGaTB have less fluorescence.
So we conducted above-mentioned experiment. We made two groups of cultured cells of Drosophila. One group had transfected BBa_K758006 and pGaTB, and the other one had transfected BBa_K758006 only. And we calculated ratios of green lighted cells to all cells on both of groups.
As results, look the following pictures.
You can detect some green cells on pictures of group that had transfected BBa_K758006 and pGaTB. Also, you can rarely detect green cells on pictures of group that had transfected BBa_K758006 only.
And we compiled statistics on the ratio of green lighted cells on pictures. In consequence, in the group transfected pGaTB, there were 356 cultured cells and there were 45 green colored cells (12.6%). And, in no pGaTB group, there were 350 cells and 2 cells were colored green (it is 0.5%).
Pictures of cultured cells transfected BBa_K758006 and pGaTB
Pictures of cultured cells transfected BBa_K758006 only
These results show that BBa_K758006 was activated by GAL4 protein and BB_K758006 expresses EGFP under condition that GAL4 protein exists.
In these circumstances, BBa_K758006 was working as expected.
Establishment of transgenic flies carrying pUAS-flag-TNFAIP3
We have injected 692 embryos (w-, delta2-3) with pUAS-flag-TNFAIP3 DNA (1mg/ml in microinjection buffer). Out of them 144 were hatched to larvae and 83 were further eclosed to adults. These 83 adult flies were crossed with yw flies and their progeny flies were inspected to identify successfully transformed w+ red eye flies. The red eye screening is still ongoing. However, we have identified six transformant strains that are listed below. Chromosomal linkage of the transgene is now under investigation.
Strain 7
Strain 10
Strain 13
Strain 14
Strain 23
Strain 29
Strain 56
Strain 65
