CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

The overall goal of this electroporation-based in vivo gene transfer approach is to evaluate the roles of genes of interest in differentiation of cerebellar granule cells using CRISPR-mediated loss of function analyses. This method can help answer key questions in the developmental neurosciences and the neuro-oncologies, such as for identification of the molecules important for normal cerebellar development and brain tumor formations. The procedure of in-utero electroporation will be demonstrated by Dr.Lena Herbst a post-doc from Professor Peter Lichter's laboratory, and then the following immunohistochemistry will be performed by Dr.Weijun Feng, a post-doc from Dr.Haikun Di's laboratory.

The main advantage of this method is to endeavor as to feasibly knock out genes of interest in the cerebellar cells, using the CRISPR-cas9 technologies, instead of the generation of the combination of knock-out animals. Although this method can give insight into gene functions during normal cerebellar development, it can also be applied to other systems, such as the development of other physical brain regions and brain tumor modeling. Begin by loading a borosilicate glass capillary into a micropipette puller and pulling to create a fine tip.

Label the capillary tip with black ink for better visualization during the injection, and draw a scaling on the glass. Working under a stereomicroscope, use a ruler and sharp needle tweezers to trim the tip to a length of about six millimeters and create a sharp, one-sided beveled edge. Filter a 1%fast green stock solution through a zero-point, 22 micron filter to remove dye particles from the solution.

Mix the single-guide RNA plasmids in equal parts with the luciferase reporter plasmid to a final concentration of at least one microgram per milliliter for each plasmid. Color the plasmid solution with fast green at a final concentration of 0.5%After anesthetizing a pregnant CD-1 mouse with isoflurane, place the mouse on its back on a heating pad, and administer analgesic. Apply eye ointment to prevent eye dryness during surgery.

Fix the lens with tape and sterilize the abdomen with a disinfectant. Cover the mouse with gauze, leaving only the surgical area exposed. After making a skin incision of about two centimeters in length, locate the linea alba and use blunt-tip tissue scissors to make a slightly smaller second incision through the peritoneum.

Moisten the opened abdominal cavity with pre-warmed sterile PBS. Next, use ring forceps to carefully extract the uterine horns from the abdominal cavity. Grasp the uterine wall at the gap between the yolk sacs of two neighboring embryos and pull gently.

Avoid any pressure on the embryo while pulling it through the incision. Once the uterine horns have been extracted, aspirate approximately 10 to 15 microliters of colored plasmid solution with the prepared glass capillary. Avoid any air bubbles during this process.

Gently hold the embryo with ring forceps and locate the neck area. Slowly penetrate the dorsal hindbrain with the tip of the glass capillary and move into the fourth ventricle. Inject about one microliter of the plasmid mix.

Confirm that the dyed DNA has not leaked from the brain, and is visible as a diamond-shaped structure. Here it is most important to fill the entire region of the fourth ventricle with plasmid solution, to deliver DNA also to the distal part of the cerebellar primordium. At the same time, make sure not to cause any bleeding during that process.

Place platinum tweezers, equipped with five millimeter diameter electrode plates laterally over the head of the embryo, with the negative pole covering the ear, and the positive pole positioned at the cerebellar primordium over the uterine wall, and apply electric square pulses. When all embryos have been electroporated, carefully place the uterine horns back into the abdominal cavity, and fill with sterile PBS. Let the uterine horns slide into a natural position.

Turn off anesthesia and place the animal belly-down into a new cage to recover. Before cryosectioning, fix the cerebella overnight in five milliliters of 4%PFA in PBS per brain, at four degrees Celsius. The next day, transfer the fixed cerebella to ten milliliters of 30%sucrose in PBS per brain, and incubate overnight at four degrees Celsius.

The following day, after the tissue has sunk to the bottom of the tube, remove the brains from the sucrose, and soak up the remaining sucrose solution with a piece of cellulose filter paper. Next, immerse each cerebellum in optimal cutting temperature compound in a disposable plastic mold, and freeze on dry ice. Cut the cryogenic block into 10 micron thick sections, using a cryostat, and keep the sections at minus 80 degrees Celsius until use.

When ready to proceed to immunostaining, first dry the slides at room temperature for 30 minutes, and then wash twice for 10 minutes each time in PBS. Circle the sections with a liquid blocker pen and incubate the sections in blocking solution for one hour at room temperature. Add primary antibodies against the molecules of interest in the blocking solution, and incubate overnight at four degrees Celsius.

Wash the slides with 0.1%Triton containing PBS three times for 10 minutes each. Then add fluorophore conjugated secondary antibody diluted in blocking solution containing DAPI, and incubate the sections for one hour at room temperature in the dark. Wash the slides in PBS-T three times for 10 minutes each time, then mount the slides, and keep at four degrees Celsius until imaging.

hek293T cells stably expressing streptococcus pyogenes cas9 were transfected with sgRNA expressing and targeting plasmids. GFP expression in live cells was monitored using a fluorescent cell imager 48 hours after transfection. This image shows an affected sgRNA sequence based on GFP expression.

This image shows lack of GFP expression resulting from a non-effective sgRNA sequence. These images show immunostaining of GFP, Ki67, and p27 in cerebella from a p7-rosa26-LSL-cas9 knock-in mouse with floxed stop cassette and downstream bicistronic cas9 and EGFP sequences, that was subject to electroporation at E13.5 with a Cre plasmid expressing control sgRNA. The section is counter-stained with DAPI.

Note GFP-expressing cells in the outer granule cell layer marked by Ki67. Once mastered, this surgery can be done in less than 30 minutes per mouse. While attempting this procedure, it is important to remember to handle the embryo very carefully, to always use sharp-tipped capillaries, and to position the electrodes in the precise angle to ensure delivery of the electric pulses to the roof of the fourth ventricle.

After its development, this technique paved the way for researchers in the field of medical sciences to explore the causal factors of the neurological disorders, including the brain tumors in the mouse cerebellum.