The overall goal of this protocol, is to describe the procedures required for the successful generation of genetically modified mice by microinjection of oocytes. This protocol can help researchers and technical staff involved either in the field of mouse genesis, but also in gene targeting. The main advantage of this technique, is that it relies on the direct injection of mouse oocytes, both for random integration, but also for gene targeting, allowing the generation of genetically modified mice in as little as six weeks.
To prepare a single guide RNA, after selecting the desired two target sequences and making primers according to the text protocol, synthesis a linear DNA template by diluting the PX330 plasmid to 10 nanograms per microliter in nuclease free water. Prepare a master mix, adding the polymerase at the end, and keep on ice. Then mix and split the solution into eight PCR tubes.
Add one microliter of PXR330 per tube, and run the following program. Next, use a PCR purification kit, a manifold and a vacuum source to purify the PCR product. Add five volumes of binding buffer to one volume of the PCR sample and mix.
Place the silicon membrane spin column on the manifold. To bind DNA, apply all eight samples to the column, in vacuum. To wash the sample, add 0.75 milliliters of wash buffer to the column and vacuum.
Then, centrifuge the column at 12, 000 times G for 60 seconds to eliminate residual ethanol. Place the column in a clean 1.5 milliliter micro centrifuge tube, then, to elute the DNA, add 30 microliters of nuclease free water to the center of the membrane. Let the column stand for one minute and centrifuge it, 12, 000 times G for 60 seconds.
Then use a spectrophotometer to measure the DNA concentration. To carry out in vitro transcription using a t7 RNA synthesis kit, prepare the master mix and incubate the reaction in a thermo cycler at 37 degree celsius for three hours. Add 28 microliters of nuclease free water and two microliters of DNAse one, and incubate the reaction for another 15 minutes.
To purify the RNA, dissolve the powder contained in the provided spin column and 650 microliters of nuclease free micro injection buffer. Carefully removing all the air bubbles, cap the tube and hydrate the solution at room temperature for five to 15 minutes. Remove the blue cap at the bottom and place the column in a two milliliter tube.
Then centrifuge the tube at 750 times G at room temperature for two minutes. Then place the column in a fresh 1.5 milliliter tube and apply 50 microliters of the RNA solution drop wise to the center, without touching the column wall. Then, spin the column at 750 times G for two minutes.
Measure the RNA concentration and assess the quality of the RNA according to the text protocol. Using nuclease free micro injection buffer, dilute the case nine mRNA to 50 nanograms per microliter and the sgRNAs to 12.5 nanograms per microliter for gene knockout experiments. Add the donor template at a concentration of 200 nanograms per microliter, for genome editing based on homology direct repair and keep it on ice.
Use the aspirator mouth piece, to wash the oocytes with four fresh drops of casein amino acids. And transfer them to a final drop of casome eight medium, overlaid with mineral oil. To carry out pro nuclear injection for random integration, under the stereo microscope, use the aspirator mouth piece to transform approximately 50 eggs to a drop of m2 medium, overlaid with mineral oil that will be used as the injection chamber.
Transfer the injection chamber to an inverted microscope and inject the fertilized oocytes with a few picoliters of the injection mix. The pronucleus will swell if the injection is successful. Perform cytoplasmic injection for gene targeting, use a mouthpiece to transfer approximately 50 eggs to a drop of casome A, containing five micrograms per milliliter cytochalasin B and incubate the eggs at 37 degree celsius and 5%carbon dioxide for five minutes.
Transfer the eggs to the injection chamber, then, at very low pressure, inject a few picoliters of the injection mix into the cytoplasm. Using the compensation pressure of the automated microinjector where possible. After the injection, use the mouthpiece to transfer the oocytes back into a drop of casomeamino acids.
Keep them at 37 degree celsius and 5%carbon dioxide until they are loaded for re-implantation into the oviduct of the pseudo pregnant females. After anesthetizing a plugged female mouse according to the text protocol, to carry out re-implantation under sterile conditions, expose the reproductive tract by using a scalpel to a one centimeter long incision parallel to the dorsal mid line. Then, with scissors, cut the muscle and use forceps to grab the fat pad.
Gently pull the ovary out, until its attached oviduct and uterus are clearly visible. Then use a vessel clamp to fix the fat pad. Under the stereo microscope, use a pair of micro scissors to make an incision into the wall of the oviduct, a few millimeters upstream of the ampulla.
Also, under the stereo microscope, load 25 micro injected eggs into the glass capillary connected to the mouthpiece. Then, insert the glass capillary into the oviduct and expel the eggs until an air bubble is visible inside the ampulla. To ensure that the re-implantation has been successful, you must check for the presence of the air bubble in the ampulla, which guarantees that all the eggs have been expelled at the right location and none remain in the glass capillary.
Gently remove the glass capillary and place the reproductive tract back into the abdomen. Then use three zero, non-absorbable surgical sutures to suture the incision and then, use wound clips to close the skin. Place the mouse on a warm mat to avoid hypothermia, and inject analgesic subcutaneously.
Monitor the mouse until a full recovery. Finally, carry out genome typing and sequencing according to the text protocol. This figure shows analytical gels demonstrating the quality of the transgene purification, and the sgRNA synthesis which are critical to successfully generate genetically modified mice.
Shown here, is a typical read out of a micro injection session for random integration. The genotyping primer should sit on the transgene and should be designed to generate a fragment of 200 to 800 base pairs. In this read out for gene targeting, the primers for selected to hybridize with the genomic DNA outside of the region eventually excised by the two guys.
This strategy allows for the direct identification of heterozygous and homozygous knock out founders. As illustrated here, when each step of the protocol is optimized, the percentage of transgenic pup should reach 10%to 25%for random integration, which is somewhat low compared to the ability of the CRISPR components to edit the mouse genome. Using CRISPR for gene targeting, the number of founders is generally higher, ranging from 25%to 100%it is not uncommon to obtain an entire progeny that is successfully homozygous when edited using CRISPR.
After its development, this technique allows researchers in fields such as, neuroscience or cancer for instance, to use new mask models to discover pathological mechanisms and eventually translate this into therapeutic strategies.
