This method could check guide RNAs in embryos, test early developmental questions, or create edited mice. Any advances in CRISPR Cas9 could be used with this method. The main advantage to this method is that it is easy to learn and uses reagents that are available to labs that have access to mice.
In the future, this technique would be useful for correcting a disease or mutation in the offspring of a companion animal or agriculture-important animal. This method is best suited for generating mouse models. In our lab, we've been using variations of this method to investigate the events surrounding fertilization and early preimplantation development, specifically, gene regulation in embryo potency.
The most challenging part is using the glass needle to move embryos from plate to plate. Demonstrating this procedure will be Chantal Diallo, a research specialist in my lab. To begin embryo collection and processing, place the euthanized female mouse on its back and spray 70%ethanol on the abdomen to be surgically opened.
To open the abdominal cavity and remove the oviducts, cut the skin layer with surgical scissors and locate the ovaries. Next, surgically remove both the oviducts, located proximal to the ovaries, and place them into individual 50-microliters droplets of M2 plus BSA medium. Under a stereo microscope, use dissection forceps to nick the ampulla of the oviduct to release the cumulus-oocyte complexes, or COCs, containing oocytes, then to avoid carryover of debris, transfer and combine each cumulus-oocyte to a single 50-microliter droplet of M2 plus BSA with a handled pipette set to 20 to 30 microliter.
Next, to remove cumulus cells from the zygotes, transfer COCs with a little extra media to a droplet of 100-microliter M2 plus hyaluronidase and gently mix by pipetting until the embryos are visibly separated from the much smaller cumulus cells. To dilute hyaluronidase and clear away loose cumulus cells, use a mouth-operated pipette to move the zygotes to a fresh 50-microliter M2 plus BSA droplet. Next, to erode the zona pellucida of the embryo and reduce additional physical obstacles for reagent delivery, transfer the embryos to a prewarmed 100-microliters droplet of acid Tyrode, or AT solution, on a 60-millimeter plate.
Continuously observe the zygotes under a stereo microscope until 30%of the zona pellucida is eroded, then to dilute the AT, pass the embryos through four 50-microliter droplets of M2 plus BSA. Next, determine an appropriate number of embryos that have been processed by counting them under the stereo microscope. Dilute BSA by passing the embryos through two droplets of 50 microliters of reduced serum media.
Next, mouth pipette 25 to 30 embryos into a 10-microliter droplet of reduced serum media, then use a handheld pipette to add 10 microliters of the ribonucleoprotein or RNP complex. Dilute the viscous glycerol from the RNP complex and homogenize the sample by pipetting 10 times or until the mixture no longer shows viscosity. Next, pipette 20 microliters of embryo and RNP mixture into a 0.1-centimeter electroporation cuvette while avoiding bubbles, then to deliver the editing reagents into the zygote, electroporate the embryos using a square-wave protocol of 30 volts, 4 to 6 pulses with 3 milliseconds pulse length, and 100 pulse intervals.
Next, add 50 microliters of M2 plus BSA into the top of the cuvette to flush the embryos from the cuvette and gently pipette out this mixture onto a plate. For embryo culturing, transfer 20 to 30 zygotes into a droplet of KSOM plus BSA in a pre-equilibrated culture plate and move the embryos to the droplet. Incubate the plate overnight at 37 degrees Celsius in 5%carbon dioxide with 95%humidity.
The following day, transfer only two-cell embryos into a fresh droplet of KSOM plus BSA mixture and return the plate to the incubator. Leave or discard the dead or unfertilized embryos. Before genotyping, wash the morula blastocyst embryos with two drops of DPBS to dilute the media components that might interfere with a PCR reaction.
Using a mouth pipette, transfer individual embryos to one well of an 8-well PCR strip and add 10 microliters of lysis buffer. Next, to lyse the embryos, program a thermal cycler to 55 degrees Celsius for 4 hours, then inactivate the proteinase K with a 10-minutes incubation at 95 degrees Celsius. In this study, an example strategy to target the mouse tyrosinase locus as a positive control, including oligo designs and genotyping strategies for non-homologous end joining and homology directed repair are shown.
When delivering a single single-guide, or sgRNA, the delivery of RNPs was up to and including 100%in the C57 black 6J and C57 black 6N mouse strains with insertion deletions formation occurring at 50 to 100%and small oligo-based replacements ranging from 17 to 63%When engineering genomic deletions, the editing effectiveness varies from 3%to 100%The embryo should be kept in as close to ideal conditions as possible so the quicker the protocol is done, the better. Also, minimize exposure to hyaluronidase and acid Tyrode's. This method describes generating an edited animal, testing implantation or gestational viability, or performing the experiment multiple times to collect measurements or images and various metrics during pre-plantation development.
