Team:BGU Israel/Results
Results
Technical Experiments
Calcium Phosphate Transfection
Title: Transfection to microglia and astrocytes via “calcium phosphate” method.
Conducted by: Sagi Angel
Date: 3-7.6.18
Aim: In this experiment we have tried to use the calcium phosphate protocol in order to transfect microglia astrocytes and HEK as control in GFP gene. The use of HEK cells as control, is due to its ability to be transfected relatively easily by the various techniques, including calcium phosphate method.
Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents and electroporation) in order to find an efficient way of inserting our plasmids into astrocytes and microglia for the continuation of the project.
Design:
[ADD PHOTO OR TABLE]
Expectations:
The expected results were the illumination of GFP under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to insert the GFP plasmid easily and the illumination would be high, and one could hope that some of the experimental cells, astrocytes and microglia, would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.
Results:
- All of the cell types survived the transfection process –no significant death rate was noticed
- In the HEK cells used as control indeed there was an expression of the GFP gene under the fluorescence microscope the expression wasn’t high
- No GFP was detected in the microglia and astrocytes cells in both option of transfection
[ADD results Photo]
Discussion:
The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transduction process worked properly, but the specific cells of the microglia and astrocytes do not respond well to the process in a manner similar to the control cells. It should be noted that the percentage of infection in the control cells was not particularly high, which may indicate a process that is not done optimally or on problems in concentrations of DNA.
Conclusion:
The process of transfusion using calcium chloride is simple and inexpensive, but does not allow high penetration rates in our target cells - astrocytes and microglia.
While there was fluorescence illumination in the control cells, it was lower than expected, which means that conditions may be improved to increase efficiency, and this may also could have effect on the target cells.
Since there are many materials that make up the buffer, it is possible that a change in quantity or lack of accurate preparation may affect the quality of the results.
It should be concluded that this method, especially in a short period of time that does not allow many adjustments and repetitions, is not suitable for the continuation of work in the project and has indeed been abandoned in favor of the use of ready-made reagents that showed higher efficiency in control cells and astrocytes and microglia.
Electroporation Transfection
Title: Knockout of IKK-β gene in microglia cell-line.
Conducted by: Einan Farhi and Mor Pasi
Date: 15.7.18
Experiment goal and significance: The objective is to create a stable cell-line of the BV2 cells with a knockout mutation of the Inhibitor of Nuclear Factor kappa-B Kinase subunit beta (IKBKB) gene. With which, we aim to demonstrate how targeting the Nuclear Factor kappa B (NFκB) pathway will diminish the amount of inflammation promoting cytokines produced by the immune representative cells of the brain.
Design:
Each transfection mixture was pipetted evenly into 6 wells of a 24 well-plate. DNA quantities that were used are as following: 2.5, 5, 9 μg of DNA. As a positive control, BV-2 cells were transfected with 5 μg pAc-GFP and as a negative control cells were electroporated with no plasmid and also seeded without electroporation. Here is a schematic diagram of the wells mentioned:
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BV2 electroporation with px601-f4/80-g2; 2.5x106 cells per cuvette; A-030 program |
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2.5 μg DNA |
2.5 μg DNA |
2.5 μg DNA |
2.5 μg DNA |
2.5 μg DNA |
2.5 μg DNA |
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5 μg DNA |
5 μg DNA |
5 μg DNA |
5 μg DNA |
5 μg DNA |
5 μg DNA |
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9 μg DNA |
9 μg DNA |
9 μg DNA |
9 μg DNA |
9 μg DNA |
9 μg DNA |
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No DNA |
No DNA |
No DNA |
No DNA |
No DNA |
No DNA |
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No transfection |
No transfection |
No transfection |
No transfection |
No transfection |
No transfection |
Expectations:
We expected to observe a GFP signal in all the wells that were successfully transfected with the px601-f4/80-g2 plasmid, while we did not expect to observe any GFP signal from the wells in the “No DNA” or “No transfection” groups.
Results:
In the following 3 days, the culture was monitored using a fluorescent microscope.
Unfortunately, no GFP positive cells were observed under the microscope lens. As the positive control showed a successful transfection in the technical sense, we concluded that there is a problem in transfecting this particular cell-line with such a large construct. This is plausible because the transfection efficiency was low even with a small construct such as pAc-GFP with only ~5% transfection efficiency (Figure1).
[add results photo]
Figure 1 - BV-2 cells, transfected with 5 μg pAc-GFP,
captured under the microscope with a green fluorescent filter.
Discussion:
As we didn’t manage to observe the GFP in the cells that were transfected with our vector, there was no reason to continue to the next step of the experiment. However, in our discussion, we arrived to an alternative explanation of No GFP result. We suggested that maybe the cells were successfully transfected and had the plasmids in them but maybe the F4/80 promoter was too weakly expressed so that a GFP was below our detection limit.
Conclusion:
The experiment with our plasmid px601-f4/80-g2 (and the rest of our plasmids with the same goal but with different guide sequences) failed. We concluded that continuing with the attempted method and plasmid would be time consuming and we would have to check for many different reasons as of why did the transfection failed. Therefore we made a change in our course of action and decided to try to accomplish the inhibition of the NFκB pathway with a different approach using Lentiviral vectors.
Transfection by transfection-reagent
Title: Transfection of the C8-D30 astrocytes and BV-2 microglia cell lines with PUC-GFP vector.
Conducted by:Liat Tsoran and Ori Tulchinsky
Date:
Aim: In this experiment we have tried to use jetPEI-Macrophage and jetPRIME transfection reagent in order to transfect microglia cells (BV2) and astrocyte cells (C8-D30), respectively, while using HEK-cell as a positive control. The use of HEK cell-line as a positive control, is due to its transfectability by the various techniques.
Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents, calcium phosphate and electrophoresis) in order to find an efficient way of inserting our plasmids into microglia for the continuation of the project.
Design:Experiment 1 – Microglia (BV2) transfection:
[add photo]
Experiment 2 – Astrocyte (C8-D30) transfection:
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Expectations:
The expected results were to detect GFP fluorescence under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to harbor the GFP containing plasmid and fluorescence would be high. One could have hoped that some of the cell-lines of our experimetns would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.
Results:
- All the cell types survived the transfection process –no significant death rate was noticed.
- In the HEK cells used as control indeed there was an expression of the GFP.
- No GFP was detected in the microglia nor the astrocyte cells in all groups of transfection.
Discussion:
The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transfection process worked properly, although that microglia and astrocytes did not respond well to the process in a manner similar to the control cells.
Conclusion:
The process of transfection using jetPEI-Macrophage transfection reagent for microglia cells and jetPRIME for astrocyte cells was unsuccessful. For this reason, we switched to other methods - prior to electrophoresis and when this method was not successful we transferred all our vectors to Lantivirus vectors and we used the Lentivirus infection method.
Microglia Experiments
Cytokines Inhibition Assay
Title: Validation of IKKB knockdown through measurement of cytokine TNFa and IL1a expression in BV2 cells.
Conducted by: Avital Bailen and Daniel Deitch.
Date: 20.9.18-11.10.18
Aim: Quantify the expression of IL1a and TNFa through qPCR using cell lines infected with the shIKK vector using a Lenti-virus packaging.
Importance: We predict that reducing the cytokines, IL-1α and TNF-α, will prevent the creation of new Reactive Astrocytes in the brain (Liddelow et al.). We predict that upon microglial IKKb knockdown reduction in the secretion of the mentioned cytokines will occur thus new reactive astrocytes formation will be prevented, this way, preventing further damage to motor neurons.
Experimental Design:
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2hr LPS |
No LPS |
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+shIKK |
No treatment |
+shIKK |
No treatment |
No cDNA |
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
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IL1 |
A |
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B |
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TNF K |
C |
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D |
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B-Actin |
E |
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F |
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+shIKK - BV2 microglia cells infected with shIKK
-shIKK -BV2 microglia cells which underwent the infection process without a plasmid
No treatment – WT BV2 microglia
Expectations:
We expect to see a lower result for IL1a and TNFa in the +shIKK, +LPS samples (columns 1,2,3) when compared to the no treatment, +LPS samples (columns 4,5). This would indicate that the infection was successful and that IKKb was indeed knocked down, leading to a decrease in cytokine secretion. We also expect to see a lower expression of cytokines in the -LPS wells (columns 6-10) when compared to the same treatments with LPS (columns 1-5). The no cDNA column should show no result, as there is no cDNA present to amplify. This column acts as a negative control in order to identify a contamination, if one occurs. Β-actin acts as the housekeeping gene, therefore we expect to see similar expression levels in all samples 1-10.
Results:
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2hr LPS |
No LPS |
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+shIKK |
No treatment |
+shIKK |
No treatment |
No cDNA |
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
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IL1a |
4.70 |
3.33 |
5.36 |
2.89 |
2.58 |
0.61 |
1.19 |
0.64 |
0.00 |
NA |
NA |
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TNF a |
22.83 |
12.80 |
15.70 |
34.53 |
43.90 |
4.27 |
3.87 |
6.33 |
1.00 |
0.50 |
NA |
[Add Bar Graph]
Discussion:
The IL1a results were not reliable. We noticed a high disparity in the given values and Cq curves without the exponential shape. As this experiment was a last attempt, due to time constraints, to quantify IL1a after many repetitions and using two different primers, we decided to exclude this cytokine from further experiments and continue exclusively with TNFa.
The TNFa results agreed with our expectations. There is a clear reduction of TNFa between the +LPS +shIKK samples when compared to the +LPS, no treatment samples. This indicates that the shIKK vector successfully induced knockdown of the IKKb gene. These values were later used with several repetitions to create the graphs shown below.
We also see lower TNFa expression in each No LPS sample when compared to the same sample with LPS. This indicates that our microglia activation process is indeed successful.
Experiment Weaknesses:
- We are missing a negative control of BV2 microglia which underwent the infection process without the plasmid. Without this sample our conclusions are less concrete as we are not able to quantify the effect of the infection process on the cytokine secretion.
- Not enough biological repetitions for all samples.
Experiment Strengths:
- There was no contamination in this experiment and the technical and biological repetitions indicated no technical errors.
Astrocyte Experiments
Astrocyte Activation
Title: Activation of astrocyte cell line C8-D30 using lipopolysaccharide (LPS) and pro-inflammatory cytokines.
Conducted by: Mor Sela
Date: 29.7.18-2.8.18
Aim: Activation of astrocytes was performed to confirm that our C8D30 astrocyte cell line can accurately model resting and reactive astrocytes for our experimental design.
Importance:Our project is based on the assumption that reactive astrocytes are a main factor in ALS and therefore our product is designed to specifically disarm them. Without a “reactive astrocyte” experimental group, we can not test the efficiency and specificity of our product in reactive astrocytes when compared to other cells in the system.
Design:
Experiment 1 – Measurement of C3 in activated astrocytes using ELISA
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C8D30 who grow with ACM + LPS (from microglia plate) |
C8D30 who grow with MCM + LPS (from microglia plate) + ACM |
C8D30 who grow with MCM (from microglia plate) without LPS + ACM Negative control |
MCM without Microglia cells + LPS (from microglia plate) + ACM + Negative control |
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V |
V |
V |
V |
Biological repetition 1 |
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V |
V |
X |
X |
Biological repetition 2 |
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X |
V |
X |
X |
Biological repetition 3 |
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X |
V |
X |
X |
Biological repetition 4 |
Expectations:
- In negative control 1 the astrocytes were treated with microglia growth medium which did not contain microglia. Then the astrocytes were grown with this medium and with astrocyte growth medium. We expected a negligible C3 concentration in negative control 1.
- In negative control 2 the astrocytes were treated with medium from microglia cell culture (without LPS). Then the astrocytes were grown with this medium and with astrocyte growth medium. We expected a negligible C3 concentration in negative control 2.
- In sample 1 the astrocytes are treated with medium from microglia cell culture with LPS. Then the astrocytes are grown with this medium and astrocyte medium. We expect a high C3 concentration in sample 1.
- In sample 2 the astrocytes are treated with medium from microglia cell culture grown in astrocyte medium with LPS. Then the astrocytes are grown with this medium and more astrocyte medium. We expect a high C3 concentration in sample 2, possibly a different concentration than seen in sample 1 as the microglia are not grown in medium specific to them.
Results:
The calibration curve is close to linearity with an R2 value >0.99, meaning that the extrapolated results, such as the unknown protein concentration, are statistically reliable.
The results show a high concentration of C3 in samples 1 and 2. There is no significant difference in C3 concentration between these two samples.
Conversely, a low C3 concentration were found in negative control 1 and 2. There is a small increase in C3 in sample 2 over sample 1.
[Add ELISA results photos]
Discussion:
This is the first attempt at activating astrocytes and was performed with LPS added to microglia medium. The purpose of this experiment was initial examination of the reactivity level of C8D30 astrocyte cell line.
The results agree with our expectations. The C3 concentration is lower in the negative controls as opposed to the astrocytes which were activated. These results indicate that the astrocytes were indeed activated and that we can create two experimental groups – resting and reactive astrocytes.
We must note that the negative controls contained a low concentration of C3, possibly because astrocytes secrete a certain amount of C3 when in a resting state. The difference in C3 concentration displayed in the negative controls does not concur with our expectations. We expected there to be no difference LPS (rather than cytokines) is not supposed to activate astrocytes, and therefore the C3 concentrations should be low and similar in both negative controls. However, this can be explained by the lack of biological repetitions. As only one biological repetition was performed for the negative controls, we can not be sure there was indeed a significant difference in concentration between them.
Experiment Weaknesses:
- We are missing a negative control of astrocytes grown in their medium with no treatment. Without this sample we can not make a reliable comparison of C3 between activated and resting astrocytes.
- Not enough and unequal biological repetitions for all samples.
- Missing additional comparative parameters such as astrocytes activated using commercial cytokines, incubation for different periods of time, etc…
- The ELISA kit is very expensive; therefore, we were limited in the repetitions we could perform for this experiment.
Experiment Strengths:
- ELISA is a very precise, sensitive method which produces quantitative results increasing the reliability of these results.
Timp1 and Steap4 Promoter Assay
Title: Promoter assay for Timp1 and Steap4 promoters in reactive astrocytes.
Conducted by: Nitzan Keidar and Mor Sela
Date: 24.9.18-28.9.18
Aim: Our goal in this experiment is to assess the strength and the specificity of the promoters Timp1 and Steap4 by quantifying the amount of luminescence produced by the Luciferase enzyme cloned downstream of these promoters, under our experimental conditions.
Importance: Our project is based on the assumption that reactive astrocytes can be targeted based on specific genetic markers (e.g Timp1 and Steap4). Non-specific expression can lead to off target activity such as healthy resting astrocytes, microglia or other neighboring brain cells.
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Experiments |
Protocols |
Notebook |
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Promoter assay in reactive astrocytes |
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Theoretical background:
“Reactive astrocytes" change their gene expression profile relative to quiescent astrocytes. Two such distinguishing genetic markers are Steap4 and Timp1 genes, expressed exclusively in reactive astrocytes1-4. Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology.
Our promoter assay kit is a "Dual-Luciferase® Reporter Assay System" of Promega. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system.
Typically, the “experimental” reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by differences in cell viability or transfection efficiency.
Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing external influences.
We used pGL3 series of firefly and Renilla luciferase vectors for the DLR™ Assay Systems. Our vectors are:
[Add picture of experiment plasmids]
Procedure:
- Co-transfect cells with plasmids pGL3+Timp1/ PGL3+Steap4 and Renilla+T7
- Allow translation of Luciferese enzyme (48 hours).
- Cell lysis to release Luc enzymes
In luminometer:
- Provide enzymes with substrate and co-factors to produce light.
- Measure light emission against controls. Renilla Luc correspond to efficiency of transfection, Firefly Luc correspond to strength of promoter.
[picture of experimental procedure]
Design:
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1. pGL3 + Timp1 & Renilla |
2. pGL3 + Timp1 & Renilla |
3. pGL3 + Timp1 & Renilla |
4. Puc GFP |
5. Puc GFP |
6. |
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7. pGL3 + Steap4 & Renilla |
8. pGL3 + Steap4 & Renilla |
9. pGL3 + Steap4 & Renilla |
10. Enhancer E7 + Renilla |
11. Enhancer E7 + Renilla |
12. Enhancer E7 + Renilla |
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13. pGL3 no promoter & Renilla |
14. pGL3 no promoter & Renilla |
15. pGL3 no promoter & Renilla |
16. Enhancer E9 + Renilla |
17. Enhancer E9 + Renilla |
18. Enhancer E9 + Renilla |
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19. No transfection |
20. No transfection |
21. No transfection |
22. |
23. |
24. |
References:
- Zamanian, Jennifer L., et al. "Genomic analysis of reactive astrogliosis." Journal of neuroscience18 (2012): 6391-6410.
- Zhang, Ye, et al. "An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex." Journal of Neuroscience36 (2014): 11929-11947.
- Tokuda, Eiichi, Eriko Okawa, and Shin‐ichi Ono. "Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase‐linked familial amyotrophic lateral sclerosis." Journal of neurochemistry1 (2009): 181-191.
- Lorenzl, S., et al. "Tissue inhibitors of matrix metalloproteinases are elevated in cerebrospinal fluid of neurodegenerative diseases." Journal of the neurological sciences1-2 (2003): 71-76.