Team:KIT-Kyoto/Project

From 2012.igem.org

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<div id="Results"><h2>Results</h2>
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<div id="Results">
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<h2>Results</h2>
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Establishment of transgenic flies carrying pUAS-flag-TNFAIP3.
+
<strong>BBa_K758006(UAS-EGFP)</strong>
<br><br>
<br><br>
-
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.
+
We transfected this plasmid to Drosophila culture cells under two different conditions. We also transfected pAct5C-GAL4 plasmids which express GAL4 proteins in a culture cell line but not in other cell line.
 +
<br>
 +
If the BBa_K758006 was assembled accurately, this plasmid expresses enough EGFP by activation of GAL4 protein. So the cell line that were co-transfected with pAct5C-GAL4 shows strong fluorescence, than the one without co-transfection with pAct5C-GAL4.
<br><br>
<br><br>
-
Strain 7
+
Then we conducted experiment mentioned above. We made two groups of Drosophila culture cells. One group had been transfected with BBa_K758006 and pAct5C-GAL4, and the other one had been transfected with BBa_K758006 alone. And we calculated ratios between the number of green lighted cells to total cells about both groups.
 +
<br><br>
 +
As results shown below, some green cells were detected among the cells co-transfected with both plasmids. But few green cells were detected among the cells transfected BBa_K758006 alone.
<br>
<br>
-
Strain 10
+
And we also compiled statistics on the ratio of green lighted cells. In consequence, in the group transfected with pAct5C-GAL4, there were 45 green colored cells (12.6%) among 356 cells in total. And, without pAct5C-GAL4, there were only 2 cells were colored green (it is 0.5%) among 350 cells in total.
 +
<br><br>
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                <img src="http://2012.igem.org/wiki/images/f/f7/WIKI-Akit.png" width="200" height="200">  <img src="http://2012.igem.org/wiki/images/c/c8/WIKI-Bkit.png" width="200" height="200">
 +
<br><br>
 +
                    Cells transfected with BBa_K758006 and pAct5C-GAL4
 +
 
 +
<br><br>
 +
                <img src="http://2012.igem.org/wiki/images/6/63/WIKI-Ckit.png" width="200" height="200">  <img src="http://2012.igem.org/wiki/images/3/31/WIKI-Dkit.png" width="200" height="200">
 +
<br><br>
 +
                       Cells transfected with BBa_K758006 alone
 +
<br><br>
 +
These results indicate that BBa_K758006 was activated by GAL4 protein and BB_K758006 EGFP expression was induced by GAL4 protein.
<br>
<br>
-
<img src="http://2012.igem.org/wiki/images/b/ba/HAEE.JPG" width="320" height="220">
+
In these circumstances, BBa_K758006 was working as expected.
<br>
<br>
-
Strain 13
 
<br>
<br>
-
<img src="http://2012.igem.org/wiki/images/a/a6/Strain13.JPG" width="320" height="220">
 
<br>
<br>
-
Strain 14
+
 
 +
 
 +
 
 +
<strong>BBa_K758005(UAS-LacZ)</strong>
<br>
<br>
-
Strain 23
+
We transfected Drosophila cells with this plasmid, too. We devided the cells into two groups. One group had been co-transfected with pAct5C-GAL4 again, and the other group had been transfected with this plasmid alone.
<br>
<br>
-
Strain 29
+
If BBa_K758005 was assembled accurately, the former group expresses LacZ strongly and the latter expresses weakly.
 +
<br><br>
 +
Generally, to test the activation of LacZ, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) is used by mesuring blue color made by the degradation of X-Gal. However, in this experiment, we conducted immunostaining by red color to test the expression of LacZ (picture F, G, I, and J). Also we use 4', 6-diamidino-2-phenylindole (DAPI) that binds and emit blue light in order to count cells (picture E and H).
 +
<br><br>
 +
As results shown below,some red cells were detected among the cells transfected with pAct5C-GAL4. They prove that LacZ is activated in this group. But the other group without transfection with pAct5C-GAL4 contains no red cells.
<br>
<br>
-
Strain 56
+
We counted cells and calculated ratios of red cells to total cells between these two cases. In consequence, by the transfection of pAct5C-GAL4, 10.1%(42/415) cells showed red color. But, without pAct5C-GAL4, no cells showed red color (0/118 ).
 +
<br><br>
 +
          <img src="http://2012.igem.org/wiki/images/b/bd/WIKI-Ekit.png"  width="200" height="200"> <img src="http://2012.igem.org/wiki/images/2/2d/WIKI-Fkit.png" width="200" height="200"> <img src="http://2012.igem.org/wiki/images/2/2f/WIKI-Gkit.png" width="200" height="200">
 +
<br>
 +
                 Drosophila cells transfected with BBa_K758005 and pAct5C-GAL4
 +
<br><br>
 +
          <img src="http://2012.igem.org/wiki/images/5/53/WIKI-Hkit.png" width="200" height="200"> <img src="http://2012.igem.org/wiki/images/1/16/WIKI-Ikit.png" width="200" height="200"> <img src="http://2012.igem.org/wiki/images/6/63/WIKI-Jkit.png" width="200" height="200">
 +
<br>
 +
                    Drosophila cells transfected with BBa_K758005 alone
 +
<br>
 +
<br>
 +
These results show that BBa_K758005 was activated and expression of LacZ were induced in the presence of the GAL4 protein.
 +
<br><br>
 +
Therfore, it can be said that BBa_K758005 was working as expected.
 +
<br><br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<strong>Establishment of transgenic flies carrying pUAS-flag-TNFAIP3</strong>
 +
<br><br>
 +
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.
 +
<br><br>
 +
Strain 7, Strain 14, Strain 23, Strain 29, Strain 56, Strain 65,
 +
<br>
 +
Strain 10
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/c/c1/HAEE2.JPG" width="320" height="220">
 +
<br>
 +
Strain 13
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/a/a6/Strain13.JPG" width="320" height="220">
<br>
<br>
-
Strain 65
+
 
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Latest revision as of 04:25, 26 September 2012




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 to Drosophila culture cells under two different conditions. We also transfected pAct5C-GAL4 plasmids which express GAL4 proteins in a culture cell line but not in other cell line.
If the BBa_K758006 was assembled accurately, this plasmid expresses enough EGFP by activation of GAL4 protein. So the cell line that were co-transfected with pAct5C-GAL4 shows strong fluorescence, than the one without co-transfection with pAct5C-GAL4.

Then we conducted experiment mentioned above. We made two groups of Drosophila culture cells. One group had been transfected with BBa_K758006 and pAct5C-GAL4, and the other one had been transfected with BBa_K758006 alone. And we calculated ratios between the number of green lighted cells to total cells about both groups.

As results shown below, some green cells were detected among the cells co-transfected with both plasmids. But few green cells were detected among the cells transfected BBa_K758006 alone.
And we also compiled statistics on the ratio of green lighted cells. In consequence, in the group transfected with pAct5C-GAL4, there were 45 green colored cells (12.6%) among 356 cells in total. And, without pAct5C-GAL4, there were only 2 cells were colored green (it is 0.5%) among 350 cells in total.

                  

                    Cells transfected with BBa_K758006 and pAct5C-GAL4

                  

                       Cells transfected with BBa_K758006 alone

These results indicate that BBa_K758006 was activated by GAL4 protein and BB_K758006 EGFP expression was induced by GAL4 protein.
In these circumstances, BBa_K758006 was working as expected.


BBa_K758005(UAS-LacZ)
We transfected Drosophila cells with this plasmid, too. We devided the cells into two groups. One group had been co-transfected with pAct5C-GAL4 again, and the other group had been transfected with this plasmid alone.
If BBa_K758005 was assembled accurately, the former group expresses LacZ strongly and the latter expresses weakly.

Generally, to test the activation of LacZ, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) is used by mesuring blue color made by the degradation of X-Gal. However, in this experiment, we conducted immunostaining by red color to test the expression of LacZ (picture F, G, I, and J). Also we use 4', 6-diamidino-2-phenylindole (DAPI) that binds and emit blue light in order to count cells (picture E and H).

As results shown below,some red cells were detected among the cells transfected with pAct5C-GAL4. They prove that LacZ is activated in this group. But the other group without transfection with pAct5C-GAL4 contains no red cells.
We counted cells and calculated ratios of red cells to total cells between these two cases. In consequence, by the transfection of pAct5C-GAL4, 10.1%(42/415) cells showed red color. But, without pAct5C-GAL4, no cells showed red color (0/118 ).

            
                 Drosophila cells transfected with BBa_K758005 and pAct5C-GAL4

            
                    Drosophila cells transfected with BBa_K758005 alone

These results show that BBa_K758005 was activated and expression of LacZ were induced in the presence of the GAL4 protein.

Therfore, it can be said that BBa_K758005 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 14, Strain 23, Strain 29, Strain 56, Strain 65,
Strain 10

Strain 13