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:
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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>

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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:

		2hr LPS										No LPS
		+shIKK			-shIKK			no treatment				+shIKK			-shIKK				no treatment				no cDNA
		1	2	3	4	5	6	7	8	9	10		11	12		13	14	15		16	17	18	19
IL1a	A
IL1a	B
TNFa	C
TNFa	D
B-Actin	E
B-Actin	F

OriginALS

Team:BGU Israel/Experiments

Technical Experiments

Calcium Phosphate Transfection

Electroporation Transfection

Transfection by transfection-reagent

Microglia Experiments

Cytokines Inhibition Assay