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Results

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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.

Design:
[ADD PHOTO OR TABLE]

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

[ADD results Photo]

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.

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:

  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:

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:

  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.

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:

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

Design:

Experiment 1 – Microglia (BV2) transfection:

[add photo]

Experiment 2 – Astrocyte (C8-D30) transfection:

[add photo]

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:

  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:

[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:

  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.

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:

 

  1. Activation of Microglia (BV2 cell line) by adding LPS to their medium inducing the secretion of cytokines7,8
  2. After 24 hours – transfer Microglia medium (cytokines +) to the “resting” Astrocyte (C8D30) wells (Activation step)1
  3. Adding commercial cytokines (IL-1a, TNF, C1q) to different “resting” Astrocyte wells (Activation step).1
  4. 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

V

V

V

V

V

V

V

Biological repetition 1

V

V

X

V

V

V

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:
  1. Sensitivity – detect protein at very low concentrations (0.1 ng protein per sample).
  2. 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
  1. Relatively cheap reagents with a long shelf life.
  2. Sensitivity and specificity higher than western blot analysis.
  3. No radiation, as opposed to exposure during binding the antibody to the protein or disposing of chemicals from western blot analysis.
  4. Faster and easier procedure than western blot analysis.
  5. The results are quantitative rather than qualitative (as in western blot analysis).
  6. 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:

  1. 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.
  2. 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.
  3. "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.)
  4. 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.
  5. 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.
  6. 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.
  7. "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
  8. "Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact." Renaud J1, Martinoli MG2.
  9. 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.‏
  10. "Advantages, Disadvantages and Modifications of Conventional ELISA" Samira Hosseini, Patricia Vázquez Villegas,Marco Rito-Palomares,Sergio O. Martinez-Chapa 31 December 2017

Timp1 and Steap4 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.

Experiments

Protocols

Notebook

Promoter assay in reactive astrocytes

 

 

 

Theoretical background:

“Reactive astrocytes" change their gene expression profile relative to quiescent astrocytes. Two such distinguishing genetic markers are Steap4 and Timp1 genes, expressed exclusively in reactive astrocytes1-4. Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology.

Our promoter assay kit is a "Dual-Luciferase® Reporter Assay System" of Promega. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system.

Typically, the “experimental” reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by differences in cell viability or transfection efficiency.

Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing external influences.

We used pGL3 series of firefly and Renilla luciferase vectors for the DLR™ Assay Systems. Our vectors are:
[Add picture of experiment plasmids]

 

Procedure:

  1. Co-transfect cells with plasmids pGL3+Timp1/ PGL3+Steap4 and Renilla+T7
  2. Allow translation of Luciferese enzyme (48 hours).
  3. Cell lysis to release Luc enzymes

In luminometer:

  1. Provide enzymes with substrate and co-factors to produce light.
  2. Measure light emission against controls. Renilla Luc correspond to efficiency of transfection, Firefly Luc correspond to strength of promoter.

[picture of experimental procedure]

 

 

Design:

1. pGL3 + Timp1 & Renilla

2. pGL3 + Timp1 & Renilla

3. pGL3 + Timp1 & Renilla

4. Puc GFP

5. Puc GFP

6.

7. pGL3 + Steap4 & Renilla

8. pGL3 + Steap4 & Renilla

9. pGL3 + Steap4 & Renilla

10. Enhancer E7 + Renilla

11. Enhancer E7 + Renilla

12. Enhancer E7 + Renilla

13. pGL3 no promoter & Renilla

14. pGL3 no promoter & Renilla

15. pGL3 no promoter & Renilla

16. Enhancer E9 + Renilla

17. Enhancer E9 + Renilla

18. Enhancer E9 + Renilla

19. No transfection

20. No transfection

21. No transfection

22.

23.

24.

 

References:

  1. Zamanian, Jennifer L., et al. "Genomic analysis of reactive astrogliosis." Journal of neuroscience18 (2012): 6391-6410.‏
  2. 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.‏
  3. 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.‏
  4. 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.‏
OriginALS

About Us


The BGU-iGEM team “OriginALS” hopes to develop an innovative therapeutic approach to prolong the life expectancy of ALS patients, using Synthetic Biology. We are dedicated to promoting ALS awareness and research in Israel through public engagement and educational activities.

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    Ben Gurion 1, Beer Sheva 8410501, Israel