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.

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:

1. Cloning of plasmid.
2. Electroporation with plasmid.
3. Validation of transfection success according to expression of GFP.
4. Validation of resulted mutation using the T7E1 assay.
5. Checking for a diminished expression of IKK-β using Western Blot analysis.
6. 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:

References:

1. 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.
2. 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.
3. Mori N and Prager D. (1996). Blood, 87, 3410 ± 3417.
4. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171, 35 ± 47.

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.

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:

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

Experimental 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:

1. 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.
2. Mori N and Prager D. (1996). Blood, 87.
3. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171.
4. 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.