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:

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:

1. All of the cell types survived the transfection process –no significant death rate was noticed
2. 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
3. No GFP was detected in the microglia and astrocytes cells in both option of transfection

Figure 1 - Cells after transfection with calcium phosphate. On top cells underregular light and on the bottom the same picture under fluorescence light. From left to right: HEK, BV2, C8D30.

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.

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.

Experiment 1 – Microglia (BV2) transfection:

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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:

1. All the cell types survived the transfection process –no significant death rate was noticed.
2. In the HEK cells used as control indeed there was an expression of the GFP.
3. 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.

Cytokines Inhibition Assay

Title: Validation of IKKB knockdown through measurement of cytokine TNFa and IL1a expression in BV2 cells.

Conducted by: Avital Bailen, Daniel Deitch, Mor Sela

Date:  20.9.18-11.10.18

Aim:  Quantify the expression of IL1a and TNFa in BV2 microglia cell line infected with the shIKK vector using Lentivirus, using qPCR for cytokines mRNA quantification and ELISA for cytokines quantification.

Importance: We predict that reducing the cytokines, IL1α and TNFα, will prevent the creation of new reactive astrocytes in the brain ¹. We predict following the microglia IKKb knockdown will reduce the secretion of the mentioned cytokines and new reactive astrocytes will not be created, preventing further damage to motor neurons.

Design:

Experiment 1: Quantify the mRNA levels of IL1a and TNFa through qPCR using BV2 cell line infected with the shIKK vector using Lentivirus.

Expectations:

We expect to see lower mRNA expression of IL1a and TNFa in the +shIKK +LPS samples (columns 1,2,3) when compared to the WT +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. Βeta-actin acts as an housekeeping gene, therefore we expect to see similar expression levels in all samples 1-10.

Results:

Discussion:

The IL1a qPCR results were not reliable. We noticed a high disparity in the given values and Cq curves without the exponential shape. As this experiment was the last attempt, out of several, to quantify mRNA levels of IL1a after 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 WT samples. This indicates that the shIKK vector successfully induced knockdown of the IKKβ gene. These values were later used with other 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 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.

Experiment 2: Quantify the expression of TNFa through qPCR using BV2 cell line infected with the shIKK vector using Lentivirus. With the addition of a negative control of BV2 microglia which underwent the infection process without the plasmid.

Design:

Expectations:

We expect to see a low expression of TNFa in the +shIKK +LPS samples (columns 1,2,3) when compared to the -shIKK +LPS (columns  4,5,6). This would indicate that the infection was successful and that IKKb was indeed knocked down, leading to a decrease in cytokine secretion. Additionally, we expect to see lower values of TNFa in the +LPS WT samples (columns 7, 8, 9) when compared to the +LPS -shIKK samples (columns 4,5,6). This would confirm that the spinfection (transfection reagent) process stresses the cells thus inducing increased cytokine secretion.

We also expect to see lower expression of cytokines in the -LPS wells (columns 10-18) when compared to the same treatments with LPS (columns 1-9).

The no cDNA column should show no result, as there is no cDNA present to amplify. This column represents a negative control in order to identify a contamination, if one occurs. Beta-actin acts as an housekeeping gene, therefore we expect to see similar expression levels in all samples 1-10.

Results:

Figure 4: Quantitative qPCR of TNFα  mRNA expression in knocked-down IKK (shIKK) microglia BV2 cells.   LPS activated BV2 microglia with and without IKK knockdown (shIKK) showed reduction in TNFα expression in microglia cells treated with shIKK compared to wild-type control cells (WT).  

The +shIKK +LPS samples expressed less TNFa than the -shIKK +LPS samples. Each sample with LPS expressed more TNFa than the same sample without LPS.

Discussion:

We were unable to include the -shIKK samples in our conclusions, as the values from these samples were erratic due to technical errors. Therefore, the results of this experiment are inconclusive. Time allowing, this experiment must be repeated.

Experiment 4: Quantify the expression of TNFa protein levels through ELISA, using supernatant of BV2 cell line infected with the shIKK vector using Lentivirus.

Design:

Expectations:

We expect to see low protein levels of TNFa in the +shIKK +LPS samples (columns 1,2,3) when compared to the -shIKK +LPS (columns 4,5,6). 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 7-12) when compared to the same treatments with LPS (columns 1-6), since non-activated microglia should secrete a negligible number of cytokines.

Results:

Figures 5-6 - ELISA for TNFα expression in knockdown IKK (shIKK) microglia BV2 cells. LPS activated BV2 microglia with and without IKKb knockdown (shIKK) showed reduction in TNFα expression in microglia cells treated with shIKK compared to -shIKK (spinfection, transfection reagent).

The +shIKK +LPS samples expressed lower TNFa protein than the -shIKK +LPS samples. Additionally,
the -LPS samples expressed TNFa protein in levels lower than the standard curve and therefore are not presented in this graph. Each sample with LPS expressed more TNFa than the same sample without LPS.

Discussion:

All of the results agreed with our expectations. There is a clear reduction of TNFa between the +LPS +shIKK samples when compared to the -shIKK +LPS samples. This indicates that the shIKK vector successfully induced knockdown to the IKKβ gene. In addition, the fact that the TNFa protein levels in the absence of induction by LPS were below the standard curve, indicates that our microglia activation process is indeed successful.

Experimental Weaknesses:

• Must preform more repetitions to statistically prove our results.

Experimental Strengths:

• Very reliable results since we are measuring protein levels.
• ELISA is a very sensitive assay

References:

1. Liddelow, Shane A., et al. “Neurotoxic reactive astrocytes are induced by activated microglia.” Nature7638 (2017): 481.

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.

Design:

Expectations:

The luminometer values from the Renilla plasmid provide an indication for the efficiency of transfection of the experimental plasmid. The Firefly luciferase values indicate the strength of the tested promoter.

The promoters of TIMP1(pTIMP1) and STEAP4 (pSTEAP4) were amplified and cloned into the pGL3 plasmid that contains the luciferase reporter gene (Luc). As the prmotoer is cloned proximal to the Luc, it allows to test the activity on the cloned promoter by the amount of luciferase expression. 

In this experiment, wells 1,2, and 3 contain reactive astrocytes co-transfected with pGL3+pTIMP1 and Renilla plasmids. According to the literature, we expect to see high levels of luminescence from the firefly luciferase^3-6.

 Wells 6, 7, and 8 contain reactive astrocytes co-transfected with pGL3+pSTEAP4 and Renilla plasmids. According to the literature, we expect to see high levels of luminescence from the firefly luciferase. This would indicate that the TIMP1 and STEAP4 promoters are expressed strongly in reactive astrocytes (C8D30 cell line)^3-6.

Wells 12, 13, and 14 contain reactive astrocytes co-transfected with pGL3 without a promoter (empty vector) and Renilla. These wells act as a negative control for this experiment. We expect to see negligible levels of luminescence from the firefly luciferase when compared to the luminescence in wells 1-3 and 6-8, as this pGL3 plasmid does not contain a promoter. The luminometer values of these wells serve as the reference for the background\noise of the experiment and will be used for calculating the relative activity for the tested promoters. 

Wells 4 and 5 act as a positive control to indicate a successful transfection, as they are transfected with Puc-GFP, a vector that can express green florescence protein (GFP). We expect see GFP expression when examining these wells in a fluorescence microscope, which would indicate a successful transfection.

Wells 9, 10, and 11 contain reactive astrocytes co-transfected with Enhancer E7 and Renilla. Wells 15, 16, and 17 contain astrocytes co-transfected with Enhancer E9 and Renilla. Enhancer E7 is an active enhancer in HEK293 that was cloned into a pGL4.23 that contains Luc and Enhancer E9 is a negative  enhancer in HEK293 cells. These wells act as a negative control for reactive astrocyte cells as the enhancers are specific for HEK293 cells, yet we have transfected them into astrocytes. Therefore, we expect low values of firefly luciferase luminescence.

Wells 18, 19, and 20 contain reactive astrocyte cells which did not undergo transfection. These wells act as a luminescence baseline for each transfected cells and to validate that our experiment is not contaminated with plasmids. We expect to see low levels of luminescence both for Renilla and firefly luciferase in these wells.

In order to increase the transfection efficiency, we preformed this experiment twice with and without trypsin. The use of trypsin should enlarge the cell surface area, therefore improving the transfection efficiency. Therefore, we used both options in order to test the promoter activity.

Results:

We found that the pTIMP1 and pSTEAP4 values are very high when compared to the negative controls of the empty vector. The values obtained from the E7 and E9 enhancers are also very low. We received reliable results both for the experiment with and without the trypsin.

Figure 9: The activity of TIMP1 and STEAP4 promoters in reactive astrocyte cell line (C8D30). Luciferase reporter assay demonstrating transcriptional activation mediated by promoters of TIMP1 and STEAP4 in C8D30 cells. The results show cells that got no treatment before transfection and cells were treated with trypsin before the transfection. The TIMP1 promoter showed 27.5-fold increased activity without trypsin and 17.07- fold increased activity with trypsin, as compared to the control (empty vector). The STEAP4 promoter showed 8.14-fold increased activity without trypsin and 9.67- fold increased activity with trypsin, as compared to the control. Enhancers 7 that serves as a negative control for Luciferase activity in C8D30 showed 4.74-fold increased activity without trypsin and 1.29- fold increased activity with trypsin, as compared to the control. Enhancers 9 that serves as a negative control for Luciferase activity in C8D30 showed decreased activity, as compared to the control. Relative luciferase expression results are presented after normalization to Renilla activity and represent the means ± standard deviation of three independent experiments.

Discussion:

As expected, the pTIMP1 and pSTEAP4 values are very high when compared to the negative controls of empty vector, which suggest that our two promoter are active in our reactive astrocyte C8D30 cell line. We also found that the promoter are active both with and without trypsin, which support our conclusion.  Additionally, the values obtained from the E7 and E9 enhancers are very low, as expected, since these enhancers are specific for HEK293 cells and not reactive astrocytes.

As we preformed this experiment on reactive astrocytes, we would be interested to test these promoters on normal resting astrocytes and after they become reactive. Therefore, our next step would be to perform this experiment on primary astrocyte cells. This way we could compare the promoter expression in resting v. reactive astrocytes and strengthen the assessment that STEAP4 and TIMP1 are specific to reactive astrocytes over resting astrocytes. We plan to preformed this experiment on mouse primary astrocytes but the plasmids that we generated are without the AAV9 elements that required for infection such plasmids into primary astrocytes, therefore, we could not do this experiment.

Conclusion:

According to the literature and this experiment, STEAP4 and TIMP1 promoters are indeed expressed strongly in our reactive astrocytes. Therefore, while working with the cell line we can rely on these promoters to express our constructs specifically in reactive astrocytes. However, further experiments with a resting astrocyte sample must be performed in order to further confirm that these two promoters are active only in reactive astrocytes, but not in resting one.

Experiment 2: promoter assay of pF4/80 in C8D30 astrocytes

As our approach is based on cell type specific activity of the promoter, we aim to test whether the pF4/80 promoter that is active promoter in microglia, will be active in astrocytes. We expected that this promoter will not be active in reactive astrocytes and only in microglia cells

Design:

Expectations:

The promoter of F4/80(pF4/80) was amplified and cloned into the pGL3 plasmid that contains the luciferase reporter gene (Luc). As the promoter is cloned proximal to the Luc, it allows to test the activity on the cloned promoter by the amount of luciferase expression. 

In this experiment, wells 1,2, 3, 4, 5 and 6 contain reactive astrocytes co-transfected with pGL3+pF4/80 and Renilla plasmids. According to the literature, we expect to see insignificant levels of luminescence from the firefly luciferase, if any This result would indicate that the F4/80 promoter is not expressed in reactive astrocytes (C8D30 cell line)¹.

Wells 7, 8, 9, 10, 11 and 12 contain reactive astrocytes co-transfected with pGL3 without a promoter (empty vector) and Renilla. These wells serve as a negative controls for this experiment. We expect to see negligible levels of luminescence from the firefly luciferase when compared to the luminescence in wells 1-6, as this pGL3 plasmid does not contain a promoter. The luminometer values of these wells serve as the reference for the background\noise of the experiment and will be used for calculating the relative activity for the tested promoters. 

Wells 13, 14, 15, 16, 17 and 18 contain reactive astrocyte cells which did not undergo transfection. These wells act as a luminescence baseline for each transfected cells and to validate that our experiment is not contaminated with plasmids. We expect to see low levels of luminescence both for Renilla and firefly luciferase in these wells.

In order to increase the transfection efficiency, we preformed this experiment twice with and without trypsin. The use of trypsin should enlarge the cell surface area, therefore improving the transfection efficiency. Therefore, we used both options in order to test the promoter activity.

Results:

We found that pF4/80  can drive luciferase expression in about 3 fold compare to the empty vector control samples, which indicate that this promoter could drive expression of gene/s.

Figure 10: The activity of F4/80 promoter in reactive astrocyte cell line (C8D30). Luciferase reporter assay demonstrating transcriptional activation mediated by promoters of F4/80 in C8D30 cells. The results show cells that got no treatment before transfection and cells were treated with trypsin before the transfection. The F4/80 promoter showed 3.05-fold increased activity without trypsin and 2.56- fold increased activity with trypsin, as compared to the control (empty vector). Relative luciferase expression results are presented after normalization to Renilla activity and represent the means ± standard deviation of three independent experiments.

Discussion:

As we found that the pF4/80 promoter is active in our reactive astrocytes cell line, it suggests that this promoter can be active both in reactive astrocytes and microglia cells.  Another explanation for the activity of this promoter could be that used murine astrocyte cell-line, when we based our experiment on the literature, where this promoter was tested on primary cells, suggesting that it might not be active in primary cells. In summary, we need to take under consideration that this promoter could be active when we use this promoter in our model system.

Experiment 3: Promoter Assay pTIMP1 and pSTEAP4 and pF4/80 in HEK293 cells.

In order to test the cell specific activity of the promoters, we plan to use HEK293 to perform the promoter assay and examine their activity. We used HEK293 cells because they are from human embryonic kidney and therefore are not relate to neuronal cells or microglia. In addition, these cells are easy to transfect which suggest that this assay will be easy to perform.

Design:

Expectations:

The promoters of TIMP1(pTIMP1), STEAP4 (pSTEAP4), F4/80 (pF4/80) were amplified and cloned into the pGL3 plasmid that contains the luciferase reporter gene (Luc). As the promoter is cloned proximal to the Luc, it allows to test the activity on the cloned promoter by the amount of luciferase expression.

In this experiment, wells 1,2 and 3 contain HEK293 cells co-transfected with pGL3+pTIMP1 and Renilla plasmids. According to the literature, we expect to see very low levels of luminescence from the firefly luciferase⁴.

Wells 4, 5, and 6 contain HEK293 cells co-transfected with pGL3+pF4/80 and Renilla plasmids. We did not have background information regarding the expression of pF4/80 in HEK293 cells but we know this gene is specific for macrophage cells, therefore we did not anticipate for pF4/80 expression in these cells.

Wells 7, 8, and 9 contain HEK293 cells co-transfected with pGL3+pSTEAP4 and Renilla plasmids. According to the literature, we didn’t expect to see any levels of luminescence from the firefly luciferase ¹.

Wells 10, 11, and 12 contain HEK293 cells co-transfected with Enhancer E7 and Renilla plasmids⁴. Enhancer E7 is an active enhancer in HEK293 that was cloned into a pGL4.23 that contains Luc. These wells act as a positive control for HEK293 cells as the enhancers are specific for HEK293 cells, Therefore, we expect a suffiient values of firefly luciferase luminescence.

Wells 13, 14, and 15 contain HEK293 cells co-transfected with pGL3 without a promoter (empty vector) and Renilla. These wells act as a negative control for this experiment. We expect to see negligible levels of luminescence from the firefly luciferase, as this pGL3 plasmid does not contain a promoter. The luminometer values of these wells serve as the reference for the background\noise of the experiment and will be used for calculating the relative activity for the tested promoters. 

Wells 16, 17, and 18 contain HEK293 cells which did not undergo transfection. These wells act as a luminescence baseline for each transfected cells and to validate that our experiment is not contaminated with plasmids. We expect to see low levels of luminescence both for Renilla and firefly luciferase in these wells.

Wells 19 and 20 act as a positive control to indicate a successful transfection, as they are transfected with Puc-GFP, a vector that can express green florescence protein (GFP). We expected to see GFP expression when examining these wells in a fluorescence microscope, which would indicate a successful transfection..

Results:

We found that the values of pTIMP1 and pF4/80 are about 2.5-4 fold when compared to the empty vector (negative control). These values are lower than enhancer 7 that is positive in HEK293 but are still show promoter activity. However, the value of pSTEAP4  is about 24 fold when compared to the  empty vector, which suggests that pSTEAP4 is a strong  promoter in HEK293 cells.

Figure 11: The activity of TIMP1, STEAP4 or F4/80 promoters in HEK293. Luciferase reporter assay demonstrating transcriptional activation mediated by promoters of TIMP1, STEAP4 and F4/80 in HEK293 cells. The TIMP1 promoter showed 2.4-fold increased activity, as compared to the control (empty vector). The STEAP4 promoter showed 24-fold increased activity, which is 10 time more that pTIMP1, as compared to the control, and the F4/80 promoter showed 4-fold increased activity, as compared to the control. Enhancer 7 that serves as a positive control for Luciferase activity in HEK293 showed 5 fold increased activity. Relative luciferase expression results are presented after normalization to Renilla activity and represent the means ± standard deviation of three independent experiments.

Discussion:

We found that these promoters are active in HEK293, but they have different activity. While TIMP1 and F4/80 showed lower activity than the positive control (enhancer 7), STEAP4 promoter showed high activity that is 10 fold from TIMP1 suggesting that this STEAP4 promoter is a strong promoter in HEK293 that can drive high expression of gene/s in this cells as well as in reactive astrocytes (previous experiment). Therefore, we can use this promoter to test our model system in HEK293 as these promoters are active and will be able to activate the required transcription in our model.

References:

1. Trakhtenberg, Ephraim F., et al. "Cell types differ in global coordination of splicing and proportion of highly expressed genes." Scientific Reports6 (2016): 32249.
2. Austyn, Jonathan M., and Siamon Gordon. "F4/80, a monoclonal antibody directed specifically against the mouse macrophage." European journal of immunology10 (1981): 805-815.‏
3. Zamanian, Jennifer L., et al. "Genomic analysis of reactive astrogliosis." Journal of neuroscience18 (2012): 6391-6410.
4. 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.
5. 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.
6. 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.‏

pMLPm Activation

Title: Validation of pMLPm activation with dCas9-VP64-gRNA and induction of apoptotic signal in HEK293 cells
Conducted by: Liat Tsoran, Mor Pasi and Ori Tulchinsky

Date: 30/9/18-14/10/18

Aim: In this experiment we wanted to validate our synthetic engineered system that should induce apoptotic death by the activation of the synthetic minimal adenovirus major late promoter (pMLPm) via dCas9-VP64-gRNA complex.

Importance: In our project, one of our goals is to engineera dCas9-VP64-gRNA system that could activate the pMLPm promoter to induce apoptotic death by the expression of exogenous reverse caspas3. Since we are using staphylococcus aureus dCas9-VP64-gRNA, we made changes in the pMLPm Protospacer Adjacent Motif (PAM), hence we had to verify pMLPm regulating activity trough the expression of the mCherry fluorophore. We would also like to verify the programmed apoptosis of the exogenous reverse caspas3 that is used in our project.

Theoretical background:

The synthetic promoter minimal adenovirus major late promoter (pMLPm) contains three repetitions of the "a1" sequence, which is complementary to the gRNA sequence, enabling dCas9-VP64-gRNA complex to target the pMLPm synthetic promoter¹. When the transcription factor VP64 (an engineered tetramer of the herpes simplex  virus VP16 transcriptional activator domain)  is fused to dCas9 enzyme it can be guided to a specific location in the genome, in this manner we can exploit it to promote downstream translation².

The dCas9-VP64-gRNA complex will target the pMLPm synthetic promoter and promote the expression of exogenous reverse caspase3, that in contrast to the endogenous caspas3 will be activated after transcription due to its autocatalytic processing, meaning this enzyme will trigger an apoptotic signal that will lead to apoptotic death  (caspase3 is responsible for chromatin condensation and DNA fragmentation)³.

mCherry reporter protein is also expressed by the pMLPm promoter, it is fused to the exogenous reverse caspase3 with Thoseaasigna virus 2A (T2A) peptide that is cleaved during translation⁴.

In order to activate pMLPm promoter and to induce apoptotic death we used 2 plasmids:

1. Lenti-viral plasmid: CMV dCas9 VP64 – in this plasmid the CMV promoter regulates the expression of dCas9 enzyme which is fused to the transcription factor VP64.
2. pSynt CMV – in this plasmid the CMV promoter regulates the expressession of the gRNA while the pMLPm promoter regulates the downstream translation of exogenous reverse caspase3 which is fused to the mCherry fluorophore.

We expected that: cells that were co-transfected with both plasmids will show mCherry expression and apoptotic death will be triggered. In order to detect leakage in pMLPm promoter we used a control of cells that were transfected only with pSynt CMV plasmid.

Design:

1. validation of pMLPm activation with dCas9-VP64-gRNA-

Plate A: FACS

sample number	1	2	3
Transfection plasmids	Lenti: CMV dCas9 VP64 + pSynt CMV	pSynt CMV	Empty plasmid
wells	A1+B1	A2+B2	A3+B3

 

Plate B: confocal microscope

sample number	1	2	3
Transfection plasmids	Lenti: CMV dCas9 VP64 + pSynt CMV	pSynt CMV	Empty plasmid
wells	A1	A2	A3

2. induction of apoptosis

Plate A:     

Plate B:       

Plate C:       

Plate D:       

Expectations:

1. Validation of pMLPm activation with dCas9-VP64-gRNA:

sample number	1	2	3
Transfection plasmids	Lenti CMV dCAs9 VP64 + pSynt CMV	pSynt CMV	Empty plasmid
mCherry expression	+	-	-

2. Induction of apoptotic signal:

Results:

Figure 12: mCherry in FACS ARIA III of HEK293 cells (excitation: 561nm emission: 610\20nm). A – cells without transfection, 78.8% of sample are viable cells. B – 48 hours post co-transfection with lent-virus:CMV-VP64 and pSynt-CMV, 88.0% of sample are viable cells.

Figure 13: Hystogramm analysis of viable HEK293 cells. Number of cells with mCherry expression. Blue - cells without transfection. Red – 48 hours post co-transfection with lenti-virus: CMV-VP64 and pSynt-CMV, 23.1% of viable cells expressed mCherry.

Figure 14: Images of mCherry in HEK293 cells 24 hours post co-transfection with lenti:CMV-VP64 and pSynt-CMV. A – bright field. B – Fluorescent microscope.

Discussion:

1. mCherry expression in HEK293 cells was validated:
• In FACS ARIA we were able to see that although we had low transfection efficiency, 23.1% of cells expressed mCherry.
• In fluorescent and confocal microscope we were able to detect mCherry fluorophore in several cells.
• Although, ew do not have a proof of apoptosis, we can see some background red-flourescene in our images, cells that undergone apoptosis will dispese their expressed m-Cerry in the media, thus the fluorescence will not be as focused as seen with localized living cells using confocal microscopy, rather there will be a background of red-flourescene, hence we may cautiously say that some of the cells that have expressed m-Cerry, have undergone apoptosis as well. This notion will require further validation.

2. When we used APC Annexin V/Dead cell apoptosis kit we run into several issus:
• The process of transfection and APC Annexin V/Dead cell apoptosis kit procedure could influence test results, since in both of these procedures cells may die.
• Co transfection efficiency has a major rule in the success of our apoptotic induction, in this experiment the co-transfection efficiency was low but we didn't had the time to repeat it.
• APC Annexin V/Dead cell apoptosis kit was calibrated to Jurkat cells. Since we are working with a different cell line further calibration is required.

Conclusion:

In this experiment we saw that pMLPm can be activated in HEK293 cells by dCas9-VP64-gRNA complex, since we were able to validate mCherry expression in several methods.

In HEK293 cells we did it when both gRNA and dCas9-VP64 are expressed by CMV promoter, our next step is to regulate pMLPm promoter activity in C8D30 cell line when dCas9-VP64-gRNA comlex is expressed by A1 reactive astrocyte specific promoters (pTimp1 and pSteap4).

In order to prove that the exogenous reverse caspase3 that was chosen in our design can trigger apoptotic death when it is regulated by pMLPm promoter, calibration on APC Annexin V/Dead cell apoptosis kit is nesseccery.

References:

1. Farzadfard, F., Perli, S. D., & Lu, T. K. (2013). Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS synthetic biology, 2(10), 604-613.‏
2. La Russa, M. F., & Qi, L. S. (2015). The new state of the art: CRISPR for gene activation and repression. Molecular and cellular biology, MCB-00512.‏
3. Srinivasula, S. M., Ahmad, M., MacFarlane, M., Luo, Z., Huang, Z., Fernandes-Alnemri, T., & Alnemri, E. S. (1998). Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits. Journal of Biological Chemistry, 273(17), 10107-10111.‏
4. Kim, J. H., Lee, S. R., Li, L. H., Park, H. J., Park, J. H., Lee, K. Y., ... & Choi, S. Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PloS one, 6(4), e18556.‏