Lentiviral vector platform have a major advantage over the most popular vector platform, specifically because of the ability of lentiviral vectors to accommodate larger genetic insert. Thus, it seems that the lentiviral vector would be the platform of choice in the application involve the delivery of CRISPR/Cas tools. This technique imitate the natural regulatory mechanism of alpha-synuclein expression which when impaired are associated with disease.
It allows fine-tuning of the expression level versus robust changes in gene expression. I would strongly recommend validating this or similar approaches using multiple guide RNAs for screening. There is great variability in guide RNA potency.
The only way to determine it is there robust and comprehensive screening and validation. The developed system can be easily adapted to other model systems such as animal models including rodent and nonhuman primate. These follow up experiment could serve as a proof of concept for future clinical studies.
Demonstrating the procedure will be Dr.Ekaterina Ilich, a senior staff member in my lab, focusing on execution of molecular cloning procedure for creating Lentiviral Vector Plasmids related to this project. She will also demonstrate the key steps involved in Lentiviral Vector production. Also demonstrating the procedure will be Dr.Lidia Tagliafierro, a postdoc from my lab focusing on the differentiation of human iPS cells into pathological relevant cells.
She will demonstrate the key steps into the establishment of assays to evaluate the methylation profiles of synuclein intron1. Ahila Sriskanda, a staff member in my lab focusing on the culture of human iPS cell-derived neural progenitor cells. To begin, develop the DNMT3A catalytic domain from the d-Cas9-DNMT3A-GFP by amplifying the DNMT3A portion of the vector.
After digesting the DNMT3A fragment using a BamH1 restriction enzyme. Cloat it into the BamH1 site of the modified PBK-301 vector carrying d-Cas9 and verified by direct Sanger sequencing, to replace the pure Meissen reporter gene and the resulting plasmid with GFP digest d-Cas9-DNMT3A-P2A pure Meissen and plasmid with FSC 1. Purify the vector fragment using a gel purification method.
Prepare the insert by digesting PBK-201 with FSC-1. Clone the FSC 1 fragment into the vector to create d-Cas9-DNMT3A-P2A-GFP. Coat 15 centimeter plates with 0.2%gelatin for the transfection and wait for 10 minutes.
Then aspirate the gelatin and add 22.5 milliliters of high glucose medium and 2.5 milliliters of previously cultured HEK-293T cell suspension. Incubate the plates at 37 degrees Celsius with 5%CO2 until reaching 70 to 80%confluence. To prepare the plasmid mix sufficient for 115 centimeter plate use the four plasmids described in the manuscript.
Add 312.5 micro liters of one molar calcium chloride to the same 15 milliliter tube with the plasmids and bring the final volume to 1.25 milliliters using sterile double distilled water. gently add 1.25 milliliters of two X BBM solution drop wise to the tube while vortexing. Incubate for 30 minutes at room temperature.
Aspirate the medium from the cells and add 22.5 milliliters of freshly prepared high glucose DMEM without serum. Add 2.5 milliliters of the transfection mixture drop wise to each plate. Swirl the plates and incubate at 37 degrees Celsius with 5%CO2 for two to three hours after incubation add 2.5 milliliters of serum per plate to achieve 10%dilution and incubate overnight at 37 degrees Celsius with 5%CO2.
To harvest the virus collect the supernatant from all the transfected cells and pull them in 50 milliliter conical tubes. Centrifuge at 400 to 450 g for 10 minutes at room temperature and then filter the supernatant through 0.45 micrometer vacuum filter unit. Next create a sucrose gradient by preparing conical ultra centrifugation tubes.
Carefully add the filtered supernatant to the gradient by equally distributing the viral supernatant among each ultra centrifugation tube filling at least three fourths of each tube. Centrifuge the samples at 70000 g for two hours at 17 degrees Celsius. Gently collect 30%to 60%sucrose fractions into clean tubes add cold 1X PBS up to 100 milliliters of total volume and pipette multiple times to mix.
Carefully add four milliliters of 20%sucrose in 1X PBS to each tube to stratify the viral preparation continue by pipetting approximately 20 to 25 milliliters of the viral solution per each tube. Carefully balance the tubes and then centrifuge at 70000 g for two hours at 17 degrees Celsius. Carefully aspirate the remaining liquid to completely remove all the liquid leaving the virus containing pellets which are barely visible as small translucent spots.
After emptying the supernatant invert the tubes on paper towels to allow the remaining liquid to drain. Add 70 micro liters of 1X PBS to the first tube and thoroughly pipette to re-suspend the pellet. Transfer the suspension to the next tube and repeat with pipetting and transferring until all pellets are re-suspended.
Add an additional 50 micro liters of 1X PBS to the first tube and mix to wash it. Then transfer the suspension to the second tube to wash and continue with washing and transferring as previously, yielding approximately 120 micro liters of the slightly milky final suspension. Centrifuge at 10000 g for 60 seconds to obtain a clear suspension transfer the supernatant to a new tube and make five micro liter aliquot and store them at negative 80 degrees Celsius place a Cryovial with MD NPC into 37 degrees Celsius heat block for two minutes and transfer the thawed cells to a 15 milliliter conical tube with 10 milliliters of warm DMEM-F12.
Centrifuge at 300 g for five minutes. Aspirate the supernatant and re-suspend the cells in two milliliters of pre warmed complete N2B2 medium. Add two milliliters of N2B27 to the cell suspension and plate two milliliters of cells in each well of the previously prepared BMM coded six well plate.
Incubate the cells at 37 degrees Celsius with 5%CO2 overnight. When MD NPCs are at 70%confluence, transduce them by adding two micro liters of lentivirus guide RNA d-Cas9-DNMT3A vectors and gently swirl the plate. After incubating the cells at the same conditions as previously for 16 hours replace the N2B27 medium 48 hours after the transduction.
Add N2B27 with five micrograms per milliliter of pure Meissen to obtain the stable MD NPC lines, grow the cells for three weeks in N2B27 plus pure Meissen to characterize the methylation profile of SNCA intron 1. First extract DNA from each stable transducer cell line using a DNA extraction kit, then use 800 nanograms of DNA to perform a bisulfide conversion with a commercially available kit and then a the bisulfide converted DNA to 20 nanograms per micro liter. For the pyro-sequencing analysis prepare the PCR master mix in a nucleus free tube transfer the reaction plate to a thermo-cycler and start the PCR.
Visualize the amplicons using two micro liters of PCR product with a 30 and bromide staining following Agarose gel electrophoresis. For pyro-sequencing assays used mixtures of un-methylated and methylated by sulfide converted DNAs and pyro-sequencing reagents as described in the manuscript. Calculate the methylation values for each CPG site using pyro-sequencing software physical yields of Lentivirus d-Cas9-DMMT3A-GFP vector and the Naive GFP vector generated using this protocol are comparable.
This suggests that the utility of the optimized vector backbone combined with the optimized production protocol results in high yields tighter of the CRISPR-dCas9 vectors transduction rates of the Lentivirus d-Cas9-DNMT3 GFP vector was four times lower than that of the Naive GFP vector suggesting that the packaging efficiency of the CRISPR d-Cas9-RNA is reduced. Transduction rates of the Naive PURO vector were five fold higher compared to the d-Cas9-DNMT3A vector. These results were further confirmed by using the Naive GFP and d-Cas9-DNMT3A-GFP.
The EB based protocol described here allows the differentiation of MD NPCs. hiPSCs expressed pleasure potency marker Oct4 while the MD NPC is expressed both Nestin and FoxA2. Seven pyro-sequencing assays were designed and validated for the quantification of the methylation status in the SNCA intron 1.
All 7 assays were validated and showed linear correlation, using the validated assays it was possible to determine the methylation levels at the 23 CPGs in the SNCA intron 1 The most important thing to remember than attending this procedure is to work with cells that show proper growth and health to ensure consistency across experiments. The production of lentil vectors highlighted in this presentation can be easily adopted to production of integrals deficient anti-viruses it's also can be easily scaled up to generate larger quantities or more concentrated vectors. This technique can pave the way to explore the pathogenic impact of genes de-regulation on age related nor the generative disorders including Alzheimer Disease and related dementias.
The advantage of lentil vectors develop here is that it's not hazard or not infectious lentil vectors have only minimal effect on cell cycle they're safe and reliable delivery platform for gene therapy applications.
