This protocol is significant because it enables visualization of not only whether an enhancer sequence can drive expression but where it can drive expression in a particular part of the body. It's fast. The injections take less than 10 minutes to complete, and whether and where a reporter gene is expressed can be seen within just a few days of sample collection.
To begin the cloning, choose or design a reporter construct. The construct used here places the enhancer candidate into the three prime untranslated region of an EGFP reporter gene expressed under the control of the hsp68 minimal promoter. To design the PCR primers for amplifying a sequence of interest and cloning into the vector, add the appropriate homology arm for Gibson cloning to the five prime end of the primers.
Using the designed primers, perform PCR from a DNA sample. Digest one to 10 micrograms of vector DNA using Pac1 and Asc1, depending on the desired number of the cloning reactions required. By employing 0.7 to 1%gel electrophoresis for 30 to 60 minutes at 100 volts, separate the restriction digest fragments.
Extract the band for the linearized vector, and purify it with a commercial gel extraction kit. Once the Gibson cloning and transformation of cells are completed, plate the cells on selective media, and incubate overnight at 37 degrees Celsius. After verifying the successful cloning of the reporter construct, inoculate five milliliters of the starter culture using the glycerol stock.
Grow the culture for eight to 16 hours in a shaking incubator at 30 degrees Celsius and 180 rotations per minute. As the broth turns cloudy, dilute the starter culture 1, 000x in 250 to 300 milliliters of fresh selective LB broth, and incubate it overnight in a shaking incubator at 30 degrees Celsius and 180 rotations per minute. Using a commercial plasmid maxi kit, purify the plasmid.
To test for the recombination of the ITRs, perform an Xma1 digestion. Visualize the bands. Ensure your digest shows the expected number of bands.
Recombination will result in fewer than expected bands. Prepare recombinant AAV mixture for delivery. If comparing the expression patterns of multiple enhancer reporter viruses, dilute each virus to equate the virus titers to the least concentrated virus.
Mix the enhancer reporter virus and constitutively expressed control reporter virus in a ratio of two to one or three to one. Add a small amount of fast green dye with a final concentration of 0.06%Break the tip of a pulled glass pipette made from a microliter-graduated capillary tube by gently piercing it into a delicate, lint-free wipe. Insert the pipette into an aspirator assembly with a pressure-applying device connected to a rubber gasket for holding a microcapillary pipette.
Draw a small volume of around 0.2 to 0.5 microliters of mineral oil into the pipette by applying negative pressure through the aspirator. Keep the aspirator assembly aside, and place the virus on ice until it is ready to inject. After cryo-anesthetizing the neonatal mice, apply negative pressure using the aspirator assembly, and draw the virus mixture into the pulled glass pipette until the meniscus passes the two-microliter tick mark.
Take the mouse away from the cold chamber, and place it on the bench top. Locate the bilateral injection sites midway between lambda and bregma, as well as the sagittal suture and each eye. Sanitize them using an alcohol wipe.
Pierce the skull of the neonatal mice using the pulled glass pipette. As the needle enters the skull, apply a positive pressure through the aspirator assembly. Dispense one microliter of the virus into the lateral ventricle, and withdraw the pipette.
Place the mouse on a heating pad or a warming chamber to recover. Return the animal to the home cage once awakened. Record fluorescence images transversing the brain section using a low magnification 5x subjective lens.
By locating the injection site, evaluate the extent of virus transduction, depending on the density and intensity of red fluorescent cells. Compare the animals that were transduced similarly. Record fluorescent images with a higher magnification objective lens of more than 25x.
Next, open the analysis software, and apply a three-by-three median filter to reduce noise. Click on the Process menu, then click on Noise, and choose the Despeckle option. Next, to subtract the background fluorescence from the images, start by separating the channels of a multi-channel image.
Click on the Image menu, then click on Color, and select the option Split Channels. Using the rectangle or circle selection tool, draw a small shape in an area of the image without any fluorescent cells. Measure the average pixel intensity of the background by clicking on Analyze and then Measure, and repeat to sample multiple times.
Average the mean gray value from five to eight background areas, and drop the decimal values. Subtract the values from each pixel in the image by selecting the Process menu, then click Math and Subtract. Next, enter the background value to subtract, and click OK.If required, merge the channels back into a multi-channel image by selecting the Image menu, then click on Color, and select Merge Channels.
To count the number of green fluorescent cells, hide the green channel, and draw a shape around the brain region expressing red fluorescence using the free-form selection tool. Click on the Measure function to measure the area, integrated density, and mean gray value of the red channel in the selected zone. Count the number of green cells by using a multi-point selection tool, and normalize the number of green cells by the integrated intensity of the red channel.
After transducing the enhancer reporter that the mouse injected intracranially at P0 with a positive AAV construct showed enhancer-driven EGFP expression in the middle lower layers of the cortex 28 days after injection. The mouse injected with a negative control construct at P0 does not show any expression of EGFP 28 days after injection, demonstrating that the enhancer elements are found to drive transcription of EGFP in the mouse brain. To visualize the successfully transduced area and control for potential variability, AAV-delivered enhancer reporter libraries of different titers were examined.
The high titer library of candidate enhancers drives broad EGFP expression while the lower titer library of candidate enhancers drives sparse CGFP expression in the P7 mouse brain. It's vital to mix the enhancer reporter AAV with the positive control at every injection in order to distinguish between enhancers without activity and failed deliveries. This procedure can be paired with immunohistochemistry, RNA FISH, or single-cell RNA sequencing to determine which cell types an enhancer is active in.
