Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Our protocols helps to create genetically-modified mice that require advanced mouse in vivo experiments, such as visualization of gene expression, genetic humanization, and conditional gene knockout. The mouse zygote genome editing technique presented here enables highly efficient, rapid, and simple induction of advanced genetic modifications, such as gene cassette knock-in and flox. Since genetically-modified mice are used in diverse research field such as neurology, immunology, metabolism, reproduction, and infectious disease, our technology presented here is fundamentally supportive this broad research field.

Demonstrating the procedure will be Natsumi Iki, Miyuki Ishida, and Yoko Tanimoto, technical staff from my laboratory. To begin, prepare two 35-millimeter Petri dishes for zygote culture and place them in the incubator at 37 degrees Celsius and 5%carbon dioxide until use. Collect the oviducts and place them in 20-microliters drop of M2 medium containing 0.75 milligrams per milliliter of hyaluronidase on the lid of the Petri dish.

Use a fine tip forceps to pick up one oviduct and perfuse about 50 microliters of M2 medium with hyaluronidase through the fimbriae. After 30 seconds, using a glass capillary, pick up morphologically normal zygotes with a small amount of medium and wash them by transferring them to fresh M16 medium droplets. Then, pool the washed zygotes into a Petri dish and repeat the process until all zygotes are retrieved.

Using the programmable pipette puller, pull some glass pipettes. Break the tip of the microinjection needle by hitting the glass ball attached to the microforge tip. Check the tip size under a microscope at 1, 000x magnification and ensure that the tip size of the microinjection needle is around one micrometer in diameter.

Use a microforge to bend the microinjection needle at about two to three millimeters from the tip. Inject 1 to 1.5 centimeters of the operation liquid into the center of the microinjection needle and attach it to the manual microinjector holder with the piezo actuator. Next, attach the holding needle to the opposite manual microinjector holder and move the operation liquid to the microinjection needle tip with gradual pressure.

Fix the manual microinjector holders on the micromanipulator. Prepare an injection chamber with three droplets. First, with 10 microliters of M2 medium, second, with five microliters of 12%polyvinylpyrrolidone in M2 medium, and third, with five microliters mixture of crRNA, tracrRNA, Cas9 protein, and the donor DNA solution.

Cover the droplets with mineral oil and set the injection chamber on the inverted microscope stage. Under the inverted microscope at a 50x magnification, lower the microinjection pipette into the 12%PVP drop. Aspirate and dispense 12%PVP to clean the inside of the microinjection needle tip and transfer it to the RNPD droplet.

Put the manual microinjector under negative pressure. Suck the RNPD solution into the microinjection needle and wait until a sufficient volume is aspirated. Under 50x magnification, fill the entire inside of the microinjection needle with liquid.

Then, stop the intake and allow the RNPD solution to be expelled gradually by setting the manual microinjector to positive pressure. Move the tip of the microinjection needle into the oil. Put 50 zygotes in the M2 droplet and gently lower the holding pipette into the same droplet.

Transfer the microinjection needle to the M2 drop containing zygotes. Switch to a 20x objective and focus on the tip of the microinjection pipette. After holding a zygote, focus on the pronucleus by moving the micromanipulator.

Insert the microinjection needle into the zygote and bring the tip close to the pronucleus. Once the tip reaches the pronucleus membrane, apply the piezo pulse to pierce. When the microinjection needle punctures the pronucleus, inject the RNPD solution and observe the swelling of the pronucleus.

Once the pronucleus is fully inflated, quickly pull out the needle and perform the same procedure on both the female and male pronuclei. Differentiate the zygotes before and after injection by moving the injected zygote to another location within the M2 droplet, and continue injecting all the zygotes present in the M2 droplet. After the injection, transfer the zygotes into a fresh M16 dish.

Select the survived zygotes 10 minutes after injection. Remove the lysed zygotes damaged by the injection and distribute survived zygotes to each droplet in the M16 dish. Place a small amount of culture medium, some air bubbles as a marker, and the zygotes in a pipette for transfer.

Using a micro shear, make an incision between the ampulla and the fimbriae of the oviduct. Introduce the zygotes into the incision and simultaneously confirm that the air bubble has been introduced. The median production efficiency of 13 independent gene cassette knock-in mice was 20.8%with a maximum of 39.5%and a minimum of 7.9%The median birth rate under this nonlethal gene targeting condition was 34.0%with a maximum of 43.3%and a minimum of 15.9%The median production efficiency of the 10 independent floxed mice was 7.7%with a maximum of 20.7%and a minimum of 2.1%Although the functions of several target genes were unknown, the median birth rate was 30.2%with a maximum of 43.8%and a minimum of 17.3%The tip diameter of the injection needle should not be too thin, and the pressure of the manual injector should be adjusted to ensure that the solution in the needle is injected and that the pronucleus is slowly inflated.