OriginALS

Experiments

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.

Experiments	Protocols	Notebook
Calcium Phosphate

Theoretical background:
Transfection of DNA into cells via calcium phosphate is a simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used with many adherent and non-adherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines.

Procedure:

The ingredients prepared according to the protocol with the GFP gene plasmid:

23 ul of PUC GFP DNA ,187 ul OF 1M CaCl2, DDW up to 750ul + 750 HEBSX2
All ingredients were made and then filtered in 0.22 filter for sterile solution
The ingredients were mixed for 30 minutes in a 1.5 ml Eppendorf for the 6 wells plate (250ul of complete reagent for each well)
In the 6 wells there were 2 options for the transformation:
a) Medium removed, 250ul reagent added. after 30 min new medium(2.5ml) added

b) 250ul reagent added + old medium(2.5ml)
After 8 hours all old medium removed and added new 2.5ml of relevantmedium

Design:
[ADD PHOTO OR TABLE]

References:

Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
Graham, F. L. and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67.
Jordan, M., Schallhorn, A. and Wurm, F. M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4): 596-601.

Electroporation Transfection

Title: Knockout of IKK-β gene in microglia cell-line.

Conducted by: Einan Farhi and Mor Pasy

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.

Experiments	Protocols	Notebook
Electroporation

Theoretical background:
In the experiment we used a px601 commercial vector designed to express a Staphylococcus aureus (SaCas9) conjugated with a Green Fluorescent Protein (GFP). Mor P. cloned the F4/80 promoter into the vector upstream of the Cas9-GFP construct instead of the original Cytomegalovirus (CMV) promoter. This promoter is considered to be expressed highly in microglia versus the other types of cells of the brain¹. Into the guide RNA sequence of the vector was cloned a targeting sequence complementary to sequences that reside in various exons of the IKBKB gene. Generally, the CRISPR/Cas9 system is used to deliver a sequence-wise accurate double strand break which should dramatically increase the chances of a knockout mutation in the targeted gene². The knockout of IKK-β should, in theory, decrease the amount of Inhibitor of kappa-B (IκB) that is sent to degradation and thus maintain a persistent inhibition of NFκB. A stronger inhibition of the NFκB complex might produce a weaker expression of its target genes, among them: Interleukin 1 subunit α (Il1α)³ and Tumor Necrosis Factor subunit α (TNFα)⁴.

Procedure:

Cloning of plasmid.
Electroporation with plasmid.
Validation of transfection success according to expression of GFP.
Validation of resulted mutation using the T7E1 assay.
Checking for a diminished expression of IKK-β using Western Blot analysis.
Cytokine assay to determine if an inhibition of the cytokine production was achieved>

[Picture of the experimental procedure will be added]

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:

BV2 electroporation with px601-f4/80-g2; 2.5x10⁶ cells per cuvette; A-030 program
2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA
5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA
9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA
No DNA	No DNA	No DNA	No DNA	No DNA	No DNA
No transfection	No transfection	No transfection	No transfection	No transfection	No transfection

References:

Helen L. Fitzsimons, Matthew J. During, CHAPTER 1 - Design and Optimization of Expression Cassettes Including Promoter Choice and Regulatory Elements, Gene Therapy of the Central Nervous System, Academic Press, 2006, Pages 3-16.
Genome engineering using the CRISPR-Cas9 system. 2013. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc. 8(11):2281-308.
Mori N and Prager D. (1996). Blood, 87, 3410 ± 3417.
Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171, 35 ± 47.

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.

Experiments	Protocols	Notebook
Transfection of the BV-2 cell-line using jetPEI transfection reagent
Transfection of the C8D30 cell-line using jetPRIME transfection reagent

Theoretical background:
jetPEI®-Macrophage allows DNA transfection of macrophages and macrophage-like cells. It contains a mannose-conjugated linear polyethylenimine that enhances binding to cells expressing mannose receptors, such as macrophages. jetPEI®-Macrophage is able to condense DNA into compact particles similarly to jetPEI®

jetPRIME® is a novel powerful transfection reagent based on a polymer formulation manufactured at Polyplus-transfection®. jetPRIME® ensures effective and reproducible DNA and siRNA transfection into mammalian cells. jetPRIME® is extremely efficient on a wide variety of cell lines. This powerful reagent only requires low amounts of nucleic acid per transfection, hence resulting in very low cytotoxicity.

Procedure:

Day 1 - Splitting of C8-D30 cells to 6-well plate for experiment
Day 2 - transfection
Day 3 - illumination of GFP under a fluorescent microscope

Design:

Experiment 1 – Microglia (BV2) transfection:

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Experiment 2 – Astrocyte (C8-D30) transfection:

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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.

Experiments	Protocols	Notebook
BV2 infection
Cytokine inhibition Assay (qPCR)
Cytokine inhibition Assay (ELISA)

Theoretical background:
The synthesis of IL1α and TNFα cytokines is mediated by the NF-kB transcription factor (Tak et al.). NF-kB activation in microglia causes motor neurons death in vitro as well as in vivo. Heterozygous inhibition of NF-kB in microglia substantially delayed disease progression in ALS mice models (Ashley et al.). In the experiment we used a commercial shikkb viral vector. This vector expresses a short hairpin RNA targeting the IKKβ mRNA. An RNA interference with the expression of IKK-β should inhibit the activation of the NFκB pathway which would produce a weaker expression of its target genes¹, among them: Interleukin 1 subunit α (Il1α)² and Tumor Necrosis Factor subunit α (TNFα)³.

Procedure:

Infect BV2 microglia cells with shIKKb plasmid.
Perform selection of transfected cells with Puromycin.
Grow BV2 cells (wild type and infected) in 6-well plates to 80% confluence.
Activate the cells with LPS for 2 hours to induce microglia activation and cytokine secretion.
Extract RNA and create cDNA.
Run qPCR.

Experimental Design:

[add table of design]

+shIKK - BV2 microglia cells infected with shIKK

-shIKK -BV2 microglia cells which underwent the infection process without a plasmid

No treatment – WT BV2 microglia

Strengths and weaknesses:

Strengths:

Sensitivity – detects synthesized product at very low concentrations.
Provides quantitative results.
Easily reproducible procedure.

Weaknesses:

As the qPCR machine is very sensitive, the experiments require a certain amount of technical ability, therefore it may take several experiments before achieving reliable results.

References:

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.
Mori N and Prager D. (1996). Blood, 87.
Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171.
Das, Amitabh, et al. “Transcriptome Sequencing Reveals That LPS-Triggered Transcriptional Responses in Established Microglia BV2 Cell Lines Are Poorly Representative of Primary Microglia.” Journal of Neuroinflammation, vol. 13, no. 1, 2016.

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.

Experiments	Protocols	Notebook
ELISA
Western Blot
Staining using Dino antigens

Theoretical background:
Reactive astrocytes are found in several forms, including forms called A1 and A2. Gene expression analysis in reactive astrocytes has shown that reactive astrocytes type A1 express many genes that are detrimental to synapses (such as complement cascade genes)¹. Studies show that A1 reactive astrocytes are produced as a result of an NFkB protein signal. A process termed nuclear factor kappa light- chain enhancer of activated B cells². Reactive astrocytes type A2 over-express neurotropic factors, which promote synapse repair. Meaning A1 reactive astrocytes are harmful while A2 reactive astrocytes are helpful to the central nervous system (CNS) and brain¹.

Recently, researchers have developed a model of exclusively A1 reactive astrocytes in cell culture which allows for targeted research. In this model, researchers quickly extract astrocytes from brain tissue which has not yet been damaged. Then these astrocytes are grown in tissue culture with the relevant medium. Finally, a cocktail of cytokines, taken from the medium of activated microglia, is added to the astrocyte culture. At this point, the astrocytes exhibit an A1 reactive astrocytes phenotype¹.

Microglia can be activated in vivo by inducing chronic CNS damage or by injection of lipopolysaccharides (LPS). Activated microglia secrete three cytokines which induce A1 reactive astrocytes: interleukin 1 alpha (IL1a), tumor necrosis factor alpha (TNFa), and complement component 1q (C1q). Adding LPS or these three cytokines in vitro produces A1 reactive astrocytes with a genetic profile very similar to A1 reactive astrocytes in vivo.¹

Experiments on this model have shown that A1 reactive astrocytes lose almost all functions displayed by resting astrocytes. A1 reactive astrocytes have very low ability to create synapses, can not perform phagocytosis, and do not induce neuronal rehabilitation or growth.

Single cell data has shown that the complement component C3 is a preferred reactive astrocytes marker to glial fibrillary acidic protein (GFAP). C3 is specific to A1 reactive astrocytes, over A2 reactive astrocytes and resting astrocytes³. Meaning A1 reactive astrocytes secrete many classical complement cascade components which accelerate synapse degeneration and other toxic substances which cause damage to neurons and oligodentrocytes^,4,5,6.

Procedure:

Activation of Microglia (BV2 cell line) by adding LPS to their medium inducing the secretion of cytokines^7,8
After 24 hours – transfer Microglia medium (cytokines +) to the “resting” Astrocyte (C8D30) wells (Activation step)¹
Adding commercial cytokines (IL-1a, TNF, C1q) to different “resting” Astrocyte wells (Activation step).¹
Validation of astrocyte reactivity - using ELISA and Western Blot to measure the expression of C3 protein in the samples⁹

[procedure img]

Design:

Experiment 1 – Measurement of C3 in activated astrocytes using ELISA

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 + C8D30 Negative control
V	V	V	V	Biological repetition 1
V	V	X	X	Biological repetition 2
X	V	X	X	Biological repetition 3
X	V	X	X	Biological repetition 4

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

ACM + 3 cytokines + C8D30 after 48hr.

ACM + 3 cytokines + C8D30 after 24 hr.

ACM + C8D30

Negative control

C8D30 who grow with ACM + LPS (from microglia plate) after 48 hr.

C8D30 who grow with ACM + LPS (from microglia plate) after 24 hr.

C8D30 who grow with MCM + LPS (from microglia plate) after 48 hr.

C8D30 who grow with MCM + LPS (from microglia plate) after 24 hr.

MCM without LPS + C8D30

Negative control

V

Biological repetition 1

V

X

V

X

Biological repetition 2

Strengths and weaknesses:

Strengths:

Adding LPS to microglia medium mimics the in vivo conditions and process better than adding commercial cytokines directly to astrocyte medium
This activation process is fast and simple.
Western Blot Analysis:

Sensitivity – detect protein at very low concentrations (0.1 ng protein per sample).
Specificity- Gel electrophoresis sorts each protein sample according to size, shape, and charge. The observed bands give an indication as to the size of the protein or polypeptide. Additionally, as the detection is based on antibody binding, the process can locate a specific protein in a sample of over 300,000 different proteins.

ELISA (Enzyme-Linked Immunosorbent Assay)¹⁰

Relatively cheap reagents with a long shelf life.
Sensitivity and specificity higher than western blot analysis.
No radiation, as opposed to exposure during binding the antibody to the protein or disposing of chemicals from western blot analysis.
Faster and easier procedure than western blot analysis.
The results are quantitative rather than qualitative (as in western blot analysis).
Applicable for a wide range of proteins.

Weaknesses:

In the article Liddelow, 2017¹ the activation is induced by injecting mouse models with LPS rather than adding LPS to cell culture. Therefore, the strength of the activation may be lower in our experiment. Additionally, we do not measure the concentration of the cytokines produced after the microglia are incubated with LPS overnight. Meaning that the astrocyte activation is induced with an unknown concentration of cytokines, as well as other unknown factors found in the medium.
Activation of astrocytes in this way does not fully mimic the process in vivo. Although there is evidence that these three cytokines are enough to induce reactive astrocytes, it is possible that other factors are involved in vivo which may affect the phenotype.
In Liddelow, 2017¹ primary astrocytes are activated, while we are working with C8D30 cell lines. Therefore, the protocol may not correspond exactly.

References:

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 activated microglia. Nature541, 481–487.
Lian, H., Yang, L., Cole, A., Sun, L., Chiang, A.C.-A., Fowler, S.W., Shim, D.J., Rodriguez-Rivera, J., Taglialatela, G., Jankowsky, J.L., et al. (2015). NFkB activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer’s disease. Neuron 85, 101–115.
"Reactive Astrocytes: Production, Function, and Therapeutic Potential" Shane A. Liddelow1,* and Ben A. Barres1,* 1Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: liddelow@stanford.edu (S.A.L.), barres@stanford.edu (B.A.B.)
Stevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N., Micheva, K.D., Mehalow, A.K., Huberman, A.D., Stafford, B., et al. (2007). The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178.
Hong, S., Beja-Glasser, V.F., Nfonoyim, B.M., Frouin, A., Li, S., Ramakrishnan, S., Merry, K.M., Shi, Q., Rosenthal, A., Barres, B.A., et al. (2016). Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716.
Sekar, A., Bialas, A.R., de Rivera, H., Davis, A., Hammond, T.R., Kamitaki, N., Tooley, K., Presumey, J., Baum, M., Van Doren, V., et al. (2016). Schizophrenia risk from complex variation of complement component 4. Nature 530,177–183.
"Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway" Xiao-jing Dai, Na Li, Le Yu, Zi-yang Chen, Rong Hua, Xia Qin, and Yong-Mei Zhang
"Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact." Renaud J1, Martinoli MG2.
Lindblom, Rickard PF, et al. "Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system." The Journal of Immunology 192.3 (2014): 1138 1153.‏
"Advantages, Disadvantages and Modifications of Conventional ELISA" Samira Hosseini, Patricia Vázquez Villegas,Marco Rito-Palomares,Sergio O. Martinez-Chapa 31 December 2017

OriginALS

Team:BGU Israel/Experiments

Technical Experiments

Calcium Phosphate Transfection

Electroporation Transfection

Transfection by transfection-reagent

Microglia Experiments

Cytokines Inhibition Assay

Astrocyte Experiments

Astrocyte Activation