This approach may be used in cell system models of endoplasmic reticulum stress by manipulating the molecular expression of downstream pair components. The main advantage of this technique is to remove the impact of paired signaling by the visualization and measurement of neural pathways. This method can contribute to understanding the molecular basis of the transition between acute and chronic phases of ER stress in diverse neurodegenerative disease models besides GM2 gangliosidosis To begin, dissect the tissue under a 20X magnifying glass with two fine-tip number five forceps, separating the frontal cortex from the meninges.
Transfer the frontal cortex to a 15-milliliter conical tube and incubate the tissue with three to four milliliters of trypsin EDTA for 15 minutes at 37 degrees Celsius for chemical digestion. After that, wash it three times with calcium magnesium ion-free Hank's balanced salt solution and 0.1%glucose. Mechanically dissociate the tissue 10 times by pipetting up and down using a 1, 000-microliter tip in DMEM plus 10%FCS.
Centrifuge the homogenate at 100 G for one minute, collect the supernatant, and complete the remaining volume in 1, 000 microliters. Plate the cell suspension on cell culture dishes at a density of 520 cells per square millimeter with two milliliters of DMEM containing 10%FCS, 1%penicillin streptomycin, and 1%of an essential amino acid additive. Incubate at 37 degrees Celsius in a humidified environment with 5%carbon dioxide for two hours.
To allow neural cell differentiation, change the medium for a serum-free neurobasal medium with the serum-free supplement. Keep cultures at 37 degrees Celsius with 5%carbon dioxide until treatment. For annealing, mix two single-stranded oligonucleotides with complimentary sequences in equal molar amounts.
Heat at 95 degrees Celsius for two minutes. Transfer the samples from the heat block and dilute the resulting product to a final concentration of 0.5-micromolar. To clone the shRNA insert into lentiviral vector pLKO.
3G, digest one micrograms of the vector with EcoR1 and PAC1 restriction enzymes. Mix gently by pipetting and incubate the reaction for three to four hours at 37 degrees Celsius. For ligation, mix the digested vector and insert it in a molar ratio of 3:1.
Incubate it with the T4 ligase and ligase buffer overnight at 16 degrees Celsius. Next, mix component E.coli with the total volume of ligation reaction and chill it on ice for 15 minutes. Place it in a 42-degree-Celsius bath for two minutes and then chill it on ice again for 15 minutes.
Transfer the total volume subjected to heat shock to a 15-milliliter tube and complete the volume to 1, 000-microliters with Luria-Bertani, or L-B liquid medium. Shake the bacteria for 90 minutes of 37 degrees Celsius in an orbital shaker at approximately 330 RPM. Centrifuge that met 5, 000 G for five minutes at four degrees Celsius.
Discard 900 microliters of the supernatant and use the remaining supernatant to resuspend the pellet. Spread bacteria suspension evenly over a solid ampicillin L-B plate and incubate for 37 degrees Celsius overnight. Pick a single colony to transfer into a 15-milliliter tube with 10 milliliters of L-B liquid medium and ampicillin.
Shake the bacteria at 37 degrees Celsius at approximately 330 RPM and centrifuge the culture to pellet the bacteria at 5, 000 G for five minutes at four degrees Celsius. After discarding the supernatant, conserve the bacterial pellet at minus 20 degrees Celsius. 24 hours before transfection, seed 1 to 5 million HEK-293 cells into 100-millimeter culture dishes.
Mix 14 micrograms of pKLO. 3G vector with a specific insert, 10.5 micrograms of packing plasmid psPAX, and 3.5 micrograms of envelope plasmid pMD2. G.Dilute 45 microliters of plasmid transfection reagent in 700 microliters of reduced-serum medium and incubate for five minutes at room temperature.
Then combine the DNA mixture with the diluted plasmid transfection reagent, mix gently, and incubate for 20 minutes at room temperature. Change the medium of the culture dish of HEK-293 cells with a fresh medium after transfection and add the entire volume of the DNA solution drop by drop. Rock the dish gently and incubate the cells at 37 degrees Celsius, 5%carbon dioxide for 48 to 72 hours to allows shRNAs to reach their optimum transduction.
Collect the medium with lentivirus and store at four degrees Celsius. Filtrate the viral supernatant with a 0.45-micrometer nylon membrane and aliquot one millimeter of the flow-through. Centrifuge at 17, 000 G for four hours.
Discard the supernatant and conserve the pellet. Allow the invisible pellet to dry, and once dried, store at minus 80 degrees Celsius. Infect the primary cortical neuron cultures with specific shRNA oligonucleotides, targeting the three-prime UTRs of either calcineurin A-alpha or CHOP and incubate them at 37 degrees Celsius with 5%carbon dioxide for one day.
After that, add GM2 stock solution to the culture dish's medium at a final concentration of two-micromolar and incubate at 37 degrees Celsius with 5%carbon dioxide for 16, 24, and 48 hours. Fix the cells with 4%paraformaldehyde and 120-millimolar sucrose in PBS for 20 minutes at 37 degrees Celsius and then wash them with PBS. Add permeabilization solution for five minutes and wash with PBS.
Use 5%BSA diluted in PBS for 45 minutes to allow blocking. Then, incubate with the anti-MAP2 antibody at four degrees Celsius overnight. After washing the cells twice with PBS at the end of the incubation time, incubate with a secondary antibody for one hour.
Mount the glasses after washing the cells with PBS. After obtaining the images with an epifluorescence inverted microscope load the picture to obtain ImageJ and subtract the background by clicking on Process followed by Subtract Background. Click on Image, Adjust, Threshold to adjust minimum values, allowing path and some tracings to be distinguishable.
Delete somas using the freehand selection. Select Traces and click on Edit, Selection, Create Selection. Get the ROI by clicking on control and T keys of the selection and conserve it.
Open the original image. Click on the ROI Manager panel and select the beginning ROI. Then click on the original image.
Once the ROI traces on the image, press control and M keys to process the statistical analysis. An attempt was made here to check whether silencing 2pERK downstream components affects the transitional phase of the unfolded protein response in the ER stress cell model. The calcineurin A-alpha gene and the CHOP gene were silenced by two specific shRNA sequences in a primary neuron cell culture for one day.
The expression is analyzed by Western blotting. A clear inhibition is observed of ER stress-mediated calcineurin A-alpha and CHOP level increase in knockdown cells. The possible link of GM2 accumulation-induced ER stress with neuronal degeneration was examined by performing MAP2 immunostaining of primary neuronal cultures.
Neurite atrophy increases significantly after inducing ER stress by incubation of GM2 at 16 to 48 hours. Silencing of calcineurin A-alpha expression significantly enhances neurite atrophy, particularly at 16 hours of GM2 accumulation relative to the GM2 untreated groups. Thus, calcineurin A-alpha knockdown accelerated the degeneration process in neurons, corroborating the pro-survival effect of calcineurin during the early phase of the unfolded protein response.
Conversely, CHOP knockdown resulted in significantly diminished neurite atrophy relative to controls precisely at 16 to 24 hours. the cell protocol with the proper primary cell culture and obtaining 90 or higher percentage of infected cells with the respective lentivirus. As the loss of neural terminal axon is the first step that we lay out in our process, it's also possible to assess this by performing assay.
This technique allows our group and others to assess some molecules as potential therapeutic tools.
