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:

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).

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.

BV2 Infection using Lentivirus

Title: Lentivirus infection of BV2 microglia cells.

Conducted by: Einan Farhi, Ori Tulchinsky, Liat Tsoran and Mor Pasi

Date:  30.8.18, 15.9.18

Aim:  The objective is to create a stable line of BV2 microglia cells with a genomic insertion of the ShIKKb vector or a stable line with the IkBM vector.

Importance: The insertion of the IκBαM or ShIKK vectors will cause knockdown to the IKKB regulation factor in the NFκB pathway. 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, microglia.

Design:

Experiment 1: BV2 spinfection with 400 µl IκBαM viral medium.

* Red wells were treated with Puromycin and Blue wells weren't

HEK293T cell line cultured in 10 cm plates was co-transfected with the IκBαM vector and 3^rd generation Lentivirus helper plasmids. Positive infection control viruses were produced in co-transfection with pGreen-puro vector and 2^nd generation Lentivirus helper plasmids. 48 hours later, viral medium was extracted, filtered and used to infect.

The BV-2 cell line was infected with the virus immediately after extraction in 24 well-plates using the spinfection method. As positive control for the infection process, we infected cells with pGreen-puro virus with which we previously demonstrated a visible expression of GFP and Puromycin resistance. As negative controls, cells were centrifuged with regular medium (no viral vector).

Results:

Wells which had cells that survived the process in them were transferred to bigger wells to grow and then RNA was extracted for qPCR analysis. We didn't manage to distinguish GFP expression in any wells that were infected with the IκBαM vector but as it was known that the expression is very weak, we went through with the following analysis. BV2 cells that were infected with pGreen-puro were GFP positive with high efficiency of infection so we concluded that the virus production and and infection were done properly in the technical sense.

Experiment 2: BV2 spinfection with 400 µl Shikkb viral medium

Design:

HEK293T cell line cultured in 10 cm plates was co-transfected with the IκBαM vector and 3^rd generation Lentivirus helper plasmids. Positive infection control viruses were produced in co-transfection with pGreen-puro vector and 2^nd generation Lentivirus helper plasmids. 48 hours later, viral medium was extracted, filtered and used to infect. Virus was concentrated using 10% Sucrose solution.

The BV-2 cell line was infected with the virus immediately after extraction in 24 well-plates using the spinfection method. As positive control for the infection process, we infected cells with pGreen-puro virus with which we previously demonstrated a visible expression of GFP and Puromycin resistance. As negative controls, cells were centrifuged with regular medium (no viral vector).

Results:

Three days after infection, selection began with 2 µg/ml of Puromycin, changing medium every other day. Wells which had cells that survived the process in them were transferred to bigger wells to grow and then RNA was extracted for qPCR analysis. BV2 cells that were infected with pGreen-puro were GFP positive with high efficiency of infection so we concluded that the virus production and infection were done properly in the technical sense.

BV2 cells infected with control virus, pGreen-Puro under the fluorescent microscope.

Conclusion:

Infection succeed according to positive control (infection of pGreen puro).

We were able to infect BV-2 cell line with Shikkb.

References:

1. ian Liang Wu, Tatsuya Abe, Ryo Inoue, Minoru Fujiki & Hidenori Kobayashi (2004) IκBαM suppresses angiogenesis and tumorigenesis promoted by a constitutively active mutant EGFR in human glioma cells, Neurological Research, 26:7, 785-791
2. Mori N and Prager D. (1996). Blood, 87, 3410 ± 3417.
3. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171, 35 ± 47.
4. Wang, X. , Li, H. , Xu, K. , Zhu, H. , Peng, Y. , Liang, A. , Li, C. , Huang, D. and Ye, W. (2016), SIRT1 expression is refractory to hypoxia and inflammatory cytokines in nucleus pulposus cells: Novel regulation by HIF‐1α and NF‐κB signaling. Cell Biol Int, 40: 716-726.

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

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.

Experiment 2 – Measurement of C3 in activated astrocytes using Western Blot

Design:

 Expectations:

• In sample 1 the astrocytes are treated with microglia medium which did not contain microglia or LPS. We expect a very faint band in the gel was these should be resting astrocytes which secrete a small amount of C3.
• In samples 2 and 3 the astrocytes are treated with medium from microglia cell culture with LPS for 24 and 48 hours, respectively. We expect very bold, thick bands with sample 3 slightly stronger than sample 2.
• In samples 4 and 5 the astrocytes are treated with medium from microglia cell culture grown in astrocyte medium with LPS for 24 and 48 hours, respectively. We expect very bold, thick bands as well. The band may be weaker than samples 2 and 3, since the microglia are not grown in their native medium and therefore may not survive as well leading to lower cytokine secretion. Alternately, the band may be stronger than samples 2 and 3 and the astrocytes grow in only their native medium and therefore will survive better and secrete more C3.
• In sample 6 the astrocytes are grown in their native medium with out any treatment. Therefore, we expect a vary faint band which will correspond to the amount of C3 secreted by resting astrocytes.
• In samples 7 and 8 the astrocytes are treated directly with TNFa, IL1a and C1q cytokines for 24 and 48 hours, respectively. We expect very bold, thick bands in these samples.

 Results:

The bands for all of the samples appear at 120kD, which is the molecular weight of the alpha subunit of the C3 protein (the long chain)¹. In some of the lanes (7-15) ,there also appear faint bands at the 45kD and 13kD height. These sizes correspond to fragments of C3 which are produced after digestion by β-mercaptoethanol (which is used during western blot analysis – adding a "Sample Buffer") .

The bands in lanes 2,3,4,5,6,7,8,14,15 present very similar color and width.

The two biological repetitions of sample 5 (lanes 9 and 10) do not match, rather they appear to have different color and width.

Lanes 11 (sample 6), 12, and 13 (sample 7) appear bolder and thicker than the rest of the bands in the gel.

[ADD WESTERN RESULTS]

Discussion:

The purpose of this experiment was to examine the level of reactivity in C8D30 astrocyte cell line. Some of the results obtained did not comply with our expectations. We expected bands 2 and 11, which correspond to the negative controls (samples 1 and 6), to appear significantly less bold than the other bands. Instead, the negative control results appear like the experimental samples. Furthermore, band 11 (astrocytes which did not receive any treatment) appears very similar to bands 12 and 13 (astrocytes activated by cytokines).

Bands 12 and 13 (astrocytes activated by cytokines) do appear as expected, as the bands are very thick and bold when compared to the negative control in band 2 (sample 1).

The difference in the band strength in lanes 2 and 11 (11 is stronger) indicates that it is preferable to grow astrocytes exclusively in their native medium, rather than in medium intended for microglia.

The different activation methods do not significantly increase the reactivity of the astrocytes. Possibly, adding the three cytokines increases the secretion of C3 protein in the astrocytes and therefore a stronger band appears in this sample (bands 12 and 13). However, the difference in band width is not significant enough to make concrete conclusions.

We repeated this experiment several times (5) with similar analysis (western blot) with similar results, therefore we conclude that these results are reliable and not results of technical or human error.

From these results we must conclude that cell line C8D30 is in a state of reactiveness, without additional induction of activation. Although this does not match the expectations which we based on Liddelow, 2017 these results are not so surprising, as the article is based on work with primary cells while we are working with cell lines.

In conclusion, we can proceed with experiments on our C8D30 astrocyte cell line as a reactive astrocyte model without performing activation treatments. The problem with these results is that we do not have a resting astrocyte model to work with.

 Experiment Weaknesses:

• We used astrocyte cell line rather than primary cells which were used in article from which we took our protocol.
• LPS was added to microglia cell line to induce cytokine secretion, rather than LPS injection to mouse models as in the article.
• Western blot analysis is less precise and sensitive than ELISA. Additionally, the results are qualitative rather than quantitative.

Experiment Strengths:

• The experiment we discussed is representative of four other similar experiments. Therefore, we base our conclusions on many repetitions which indicate that the C8D30 cell line is in a reactive state.

Future Plans:

• GFAP is also considered a marker of A1 reactive astrocytes³. Therefore, we recommend using both C3 and GFAP as markers to strengthen the reliability of experimental results.
• We recommend using ELISA rather than western blot analysis to measure reactivity.
• It is recommended to perform immunohistochemistry with markers for reactivity in the specific cell line used.

 Experiment 3 – Identification of reactive astrocytes using immuno-staining:

The previous methods showed opposing results. The ELISA results indicated that LPS activates C8D30 astrocytes, while the western blot analysis results indicated that LPS does not significantly increase the cell line reactivity. Therefore, we decided to perform a third experiment to confirm whether or not the C8D30 cell line is in a reactive state.

We organized a collaboration with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv university. The DFM lab specializes in combines cell biology, molecular biology, biochemistry, immunology, cancer research, as well as advanced genome-wide techniques and microscopy. The DFM lab performs immune-staining to confirm reactive states in glioblastoma and therefore were well equipped to assist us with this matter, as our lab does not possess the necessary equipment. The DFM lab performed immune-staining on a sample of our C8D30 astrocyte cell line.

Results:

[Add Dino results]

GFAP and Nestin, both reactivity markers, were found and appear in the same location as the cells stained with DAPI.

Discussion:

The immune-staining results confirm the results we obtained in through western blog analysis. The C8D30 cell line is always in a reactive state. Apparently, the differences in C3 concentration we found when using ELISA were negligible and would have been rejected with enough repetitions.

Conclusion:

The C8D30 astrocyte cell line is in a reactive state without any additional treatment. In order to support this conclusion, we recommend using ELISA with many biological repetitions and biomarkers.

References:

1. Third Component of Complement (C3): Structural Properties in Relation to Functions (topology of functional sites/physiologic C3 fragments/C3-membrane interaction/immune adherence/enzyme subunit) VIKTOR A. BOKISCH, MANFRED P. DIERICH, AND HANS J. MULLER-EBERHARD Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 Contributed by Hans J. Muller-Eberhard, March 10, 1975.
2. Liddelow, S.A., Guttenplan, K.A., Clarke, L.E., Bennett, F.C., Bohlen, C.J., Schirmer, L., Bennett, M.L., M€unch, A.E., Chung, W.S., Peterson, T.C., et al.(2017). Neurotoxic reactive astrocytes are induced by activated microglia. Nature541, 481–487.

3. "Reactive Astrocytes: Production, Function, and Therapeutic Potential"Shane A. Liddelow1,* and Ben A. Barres1,Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA,Correspondence: liddelow@stanford.edu (S.A.L.), barres@stanford.edu (B.A.B.),J Neurosci Methods.;1995 May;58(1-2):181-92.

pMLPm Activation

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

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:

[ADD RESULTS1 ], [ADD RESULTS 2]

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.

[Add Results 3]

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.

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

[ADD RESULTS 4]

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.‏

Apoptosis Induction

Title: Validation of pMLPm activation with dCas9-VP64-gRNA and induction of apoptosis in C8-D30 astrocyte cells
Conducted by: Liat Tsoran and Mor Pasi

Date: 30/9/18-14/10/18

Aim: In this experiment we wanted to test the activity and efficiency of 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 that is expressed by A1 reactive astrocyte specific promoters.

Importance: In the astrocyte pathway we aimed to eliminate only A1 reactive astrocyte by triggering apoptotic death, while maintaining the resting cells in the central bervous system (CNS) intact. In this experiment we wanted to test the effectiveness of our enginnered system, in which, dCas9-VP64-gRNA complex that is expressed under the regulation of A1 reactive astrocyte specific markers, can activate the pMLPm promoter to regulate the expression of exogenous reverse caspase3 that will trigger apoptosis.

Theoretical background:

pSTEAP4 (Six Transmembrane Epithelial Antigen of Prostate 4 promoter)  and pTIMP1 (Tissue inhibitor of metalloproteinases-1 promoter) are regulating genes expression which are highly expressed in A1 reactive astrocyte cells when compared to other cells in the central nervous system^1,2

In our therapeutic approach, we wanted to use these markers in order to express dCas9-VP64-gRNA complex that will activate a synthetic promoter to induce apoptosis.

The synthetic 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 caspase3 will be activated after transcription due to its autocatalytic processing, meaning that 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: pTIMP1 dCas9 VP64 – in this plasmid TIMP1 promoter express dCas9 enzyme which is fused to the transcription factor VP64.
2. pSynt – in this plasmid pSTEAP4 promoter express gRNA. And pMLPm promoter regulate the downstream translation of reverse caspase3 which is fused to mCherry fluorophore.

A1 reactive astrocyte cells that were co-transfection with both plasmids will show mCherry expression and will 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 plasmid.

Design:

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

Plate A: FACS     

sample number	1	2	3
Transfection plasmids	Lenti: pTIMP1 dCas9 VP64 + pSynt CMV	pSynt	Empty plasmid
wells	A1+B1	A2+B2	A3+B3

 

Plate B: confocal microscope     

sample number	1	2	3
Transfection plasmids	Lenti: pTIMP1 dCas9 VP64 + pSynt CMV	pSynt	Empty plasmid
wells	A1	A2	A3

 

2. induction of apoptotic signal-

Plate A:       

sample number	1	2
sample name	pTIMP1 dCas+ pSynt	pSynt
Transfection plasmids	Lenti: pTIMP1 dCas9 VP64 + pSynt	pSynt
wells	A1-3	B1-3
APC annexin V	+	+
Sytox green	+	+

 

Plate B: 

sample number	3	4
sample name	mCherry control (for FACS calibration)	annexin control (for FACS calibration)
Transfection plasmids	Lenti: pTIMP1 dCas9 VP64 + pSynt	Lenti: pTIMP1 dCas9 VP64 + pSynt
wells	A1-3	B1-3
APC annexin V	-	+
Sytox green	-	-


Plate C:       

sample number	5	6
sample name	necrosis sytox control (for FACS calibration)	necrosis
Transfection plasmids	-	-
wells	A1-3	B1-3
APC annexin V	-	+
Sytox green	+	+


Plate D:       

sample number	7
sample name	without apoptosis induction
Transfection plasmids	-
wells	A1-3
APC annexin V	+
Sytox green	+

 

Expectations:

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

sample number	1	2	3
Transfection plasmids	Lenti Timp1 dCAs9 VP64 + pSynt	pSynt	Empty plasmid
mCherry expression	+	-	-

2. induction of apoptotic signal:

sample number	1	2	3	4	5	6	7
sample name	Timp1 dcas+ pSynt	PSynt	mCherry control (for FACS calibration)	annexin control (for FACS calibration)	necrosis sytox control (for FACS calibration)	necrosis	without apoptosis induction
Dead	+	-	+	+	+	+	-
Alive	-	+	-	-	-	-	+
Necrosis	-	-	-	-	+	+	-
Apoptosis	+	-	+	+	-	-	-
mCherry expression	+	-	+	+	-	-	-

Results:

[ADD RESULTS]

Images of mCherry in C8-D30 cells 24 hours post co-transfection with lenti:pTimp1-VP64 and pSynt. A – Fluorescent microscope. B – Bright field .

Discussion:

1. mCherry expression in C8-D30 reactive astrocyte cells was visible. Demonstrating the activity of our dcas9 editing approach and verifying expression of rev-caspase3. In fluorescent microscope we were able to detect mCherry fluorophore in several cells.
2. By demonstrating mCherry expression we were able to demonstrate both the specificity of our chosen promoter as well as the ability of our system to drive the expression of caspase3 (through the mCherry marker) we have designed into our plasmid.
3. When we used APC Annexin V/Dead cell apoptosis kit we run into several issues:
• 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 role in the success of our apoptotic induction, in this experiment the co-transfection efficiency was low but we didn't have time to repeat it.
• APC Annexin V/Dead cell apoptosis kit was calibrated in 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 C8-D30 cells by dCas9-VP64-gRNA complex, since we were able to validate mCherry expression by several methods.

This means that pMLPm promoter activity can be regulated in reactive astrocytes (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.

Since co-transfection efficiency is very low, the next step is to infect the cells with lenti:pTimp1-dCAs9-VP64, and create a stable cell-line, and only only then to insert the pSynt plasmid (either by transfection or by AAV2/9 infection).

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