In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big AVV constructs that does not allow us to use a reporter gene like CRISPR based constructs. By using this method, we can simply quantify the transduction rate of AAVs in retina.
This method relies on sensitivity and absolute quantification of the digital droplet PCR and yields straight forward results. In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big area constructs that does not allow us to use a reporter gene like CRISPR based constructs.
By using this method, we can simply quantify the transduction rate of AAVs in retina. This method relies on sensitivity and absolute quantification of the two droplet PCR and yields straight forward results. Because of the efficiency of the digital droplet PCR methodology, several laboratories already switch from quantitative real-time PCR to digital droplet PCR or AAV titrating.
This method can also provide a basis for mitochondrial DNA quantification in retina, as well as checking the efficiency of the CRISPR based mutation correction for gene therapy in the mouse retina. Prepare transfection mixture with AAV helper plasmid, AAV capsid plasmid, AAV vector, and PI solution in five milliliters demand. Add the five milliliters prepared transfection mixture into culture media.
Incubate transfected cells at 37 degrees celsius. Collect the media at 48 to 60 hours post transfection. Digest the media with DNAs at a final concentration of 250 units per milliliter for 30 minutes at 37 degrees Celsius and a centrifuge it at 4, 000 g four degrees Celsius for 30 minutes.
Pre-wet the regenerated cellulose membrane with PBS. Filter the media with 0.22 micrometers syringe filter into the pre-wetted regenerated cellulose membrane for 100 kilo Daltons. Centrifuge the regenerated cellulose membrane at 4, 000 G four degrees Celsius for 30 minutes and discard the media.
Wash and centrifuge the regenerated cellulose membrane with PBS containing 0.001%pluronic F68 three times at 4000 G four degree Celsius for 30 minutes. Collect concentrated AAVs from the top part of the regenerated cellulose membrane and allocate for further use. Here we showed how to make a smaller scale AAV production.
After vetting this protocol, you should be able to make AVVs that is sufficient to see the reporter expression in the retina. You can also follow the protocol for digital PCR to be able to titer the produce AAVs. Apply one drop of 0.5%tropicamide and 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils.
Anesthetize mice with isoflurane and verify the reflexes. Apply 0.3%tobramycin and 0.1%dexamethasone sterile ophthalmic solution eye drop to each eye before the injection procedure. Use the surgical hook to stabilize the upper eyelid.
Slightly pull back to expose the dorsal part of the eye. Tape the hook to the band to hold in position. Remove conjunctiva with the curved-iris scissors to expose the sclera of the eye.
Use a fresh and sterile 30G insulin syringe to puncture the sclera. Insert the needle of the micro syringe through the same puncture using micro manipulator. Place the tip of the needle behind the lens in the middle of the eye cup.
Inject one micro liter of AAV solution slowly into the vitreous of the eye. Leave the needle in place for one minute and slowly withdraw the needle. Apply anti-inflammatory and anti-bacterial topical gel treatment containing 0.3%tobramycin and 0.1%dexamethasone to eye cup.
Apply one drop off 0.5%tropicamide. And 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils. Apply carbamide two milligrams per gram topical gel onto the surface of the eyes.
Adjust the mouse stand as needed to center the mouse eye. Perform fundus and fluorescence imaging on both eyes to follow TdTomato expression one week and two weeks after intravitreal injection. Euthanize the animals using carbon dioxide inhalation before the procedure.
Shift eye ball forward with the help of a 13.5 centimeters splitter forceps. Remove the lens together with the leftover vitreous. Cut the connection of the eyeball to the optic nerve with a precision curved forceps and gently squeeze retina with the same curved forceps.
Isolate genomic DNA with a commercialized proteinase K digest film-based tissue genomic DNA kit. Digest retina at 55 degrees Celsius 200 G for 30 minutes with proteinase K.Lyse retina completely and follow the manufacturer'sprotocol. In these protocols, we have shown intravitreal injection fundus florescence imaging, as well as retina and genomic DNA isolation.
These are very critical procedures for retina, and by following this protocol, you should be able to inject the produced AAVs and follow up their expression profile. And ladle that transduction efficiency by quantifying AAV genomes in retinal genomic DNA. Here, you can see the basic application of digital droplet PCR, where you can make an absolute quantification of AAV genome that transduce in the retinal cells.
Dilute genomic DNAs from injected retinas in 0.05%pluronic F 68 solution to reach a final concentration of one nanogram per microliter for each sample. Perform droplet generation in a 20 microliters PCR reaction mix, including one nanogram genomic DNA 125 nanomolars primers and 2X evergreen super mix. Load sample mix to the middle part of the cartridge for droplet generation.
Afterwards add 70 microliters droplet generation oil into the bottom row of cartridge. Put the gasket on top of cartridge. Make sure that there is no gap between the gasket and the cartridge.
Then place it into the droplet generator. Gently take approximately 40 microliters of droplets that are generated in the top bell. Add droplet solution to semi skirted 96-Well PCR reaction plate.
Seal the PCR plate with a PCR plate sealer by using aluminum sealing foil. Place the PCR plate into the 96-Well heat sealed thermal cycler. Use the ddPCR protocol indicated in the article.
Place PCR plate into the droplet reader to analyze data. After watching this video, you should be able to perform basic digital PCR technique. You can also use exerted cells with minor amounts of material, which is ideal for digital PCR technique.
However, you should carefully adjust the target DNA amount to not exceed the limit of digital droplet PCR. Here's the flow chart for the experimental procedure. We performed small-scale AAV production, which is followed by AAV titering.
Then we performed intravitreal injection. Afterwards, we quantified the fluorescent intensity by fundus fluorescence imaging. Then isolated the mouse retinas and extracted genomic DNA from retinas for ddPCR.
Titering AAV is also critical to be able to correctly quantify AAV transduction. ddPCR depends on generation of droplets that has diluted target AAV genom. Representative ddPCR results shows positive and negative droplets for the AVV genome.
Positive above the threshold and negative is below. After multiplying with the dilution factors, titer of AAVs were found to be 10 to the 12 in genome copies per milliliter. In order to correctly quantify AAV tranduction efficiency, several retinas were injected.
Injected animals were imaged, and fluorescence mean intensity was calculated. At two weeks time point, retinas were isolated. And ddPCR was performed using WPRE and 18 S primers.
Fluorescence intensity of AAV injected retinas were correlated with AAV genome calculations, showing the power of the methodology. Here, we have shown several techniques, including small-scale AAV preparation intravitreal injection.
