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<p>Figure 4. Complete set-up for rearing of Diaphorina citri, consisting in two mesh-lined cages and complete with light and temperature conditioning.</br></br></p> | <p>Figure 4. Complete set-up for rearing of Diaphorina citri, consisting in two mesh-lined cages and complete with light and temperature conditioning.</br></br></p> | ||
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Revision as of 07:01, 1 November 2017
![](https://static.igem.org/mediawiki/2017/0/07/TEC-CEM-home_hojas_header.png)
Results
siRNA confirmation
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Sequencing
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Blue BSLA colonies
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Real Time PCR
Since the focus of this project is to verify the effective gene silencing with the four different siRNA we designed, the direct measurement of the amount of mRNA in the cells was required. The first step was to perform a Two-Step Reverse Transcriptase PCR to find out where do the siRNA molecules hybridize with the mRNA. The results are shown below. .
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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas.
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Table: Expected products for the amplifications in the Two-step RT-PCR.
Product | Expected size |
---|---|
Tubulin | 195 bp |
Rac I | 193 bp |
WNT | 200 bp |
AWD | 180 bp |
SOD | 199 bp |
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Diaphorina citri primary cell culture
The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium A were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed.
Reagent | Medium A | Medium B |
---|---|---|
200 mM L-Glutamine | 1 mL | 1 mL |
Penicillin and Streptomycin 1000X | 1.5 mL | 1.5 mL |
Fungizone 500X | 0.2 mL | 0.2 mL |
Schneider Medium | 72 mL | 80 mL |
199 Medium 10X with Hanks’ salts | 6 mL | ----- |
Fetal Bovine Serum (heat-inactivated) | 20 mL | 20 mL |
0.06 M Histidine solution | 1 mL | ----- |
![](https://static.igem.org/mediawiki/2017/7/79/TEC-CEM_cellculture12hr.png)
Figure 1. Cell culture 12 hours after extraction.
![](https://static.igem.org/mediawiki/2017/e/e8/TEC-CEM_cellculture2day.png)
Figure 2. Cell culture 2 days after extraction, observed under an inverted microscope at 10x. No significant cell replication can be observed compared to the first day,
![](https://static.igem.org/mediawiki/2017/d/d4/TEC-CEM_cellculture2week.png)
Figure 3. Cell culture 2 weeks after extraction, observed under an inverted microscope at 10x. Cell replication can be observed.
Diaphorina citri laboratory development
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![](https://static.igem.org/mediawiki/2017/f/fe/TEC-CEM_diaphoresults1.png)
Figure 1. Detail of mesh lining the interior of the cages. The size of the mesh prevents the insects from escaping while providing light, air flow and visibility.
![](https://static.igem.org/mediawiki/2017/7/75/TEC-CEM_diaphoresults3.png)
Figure 2. Presence of dew in the plant's leaves, resulting from nymphs feeding on the plant, which indicates reproduction.
![](https://static.igem.org/mediawiki/2017/7/7b/TEC-CEM_diaphoresults2.png)
Figure 3. Detail of plant leaves after two weeks of rearing. The presence of eggs in the stems of the plant can be observed.
![](https://static.igem.org/mediawiki/2017/9/98/TEC-CEM_diaphores.png)
Figure 4. Complete set-up for rearing of Diaphorina citri, consisting in two mesh-lined cages and complete with light and temperature conditioning.
![](https://static.igem.org/mediawiki/2017/f/f4/TEC-CEM_diaphoresults0.png)
Figure 5. Presence of adult Diaphorina citri specimens after one month of rearing.