The overall goal of this protocol is to provided viewers with detailed procedures to produce Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex. This PN injection protocol we developed in the Golden Syrian Hamster makes genetic engineering in this business possible. This technology is a great contribution to the field of animal model development.
Due towards extreme sensitivity of hamster embryos to the environment, we developed a unique pronuclear injection protocol to accommodate the special niche of hamster embryos. The development of this technique is a great step forward in human disease modeling, because for the first time, genetically engineered hamster models can be produced. For this experiment, one day prior to pronuclear or PN injection, prepare Zygote handling dishes and PN injection drop in a lid of a 35 millimeter dish with HECM-9 medium.
Cover the medium drops with mineral oil. To balance the drops and HECM-9 medium, incubate it at 37.5 degrees celsius overnight. On the day of PN injection, prepare embryo-flushing dishes using HECM-9.
Store them in the incubator until needed. To isolate oviducts, place a euthanized superovulated female hamster in dorsal recumbency on a surgical drape. Scrub the abdomen with 70%ethanol.
Next, firmly hold skin and abdominal muscle layer at the midline, and make an incision along the end of the abdomen. To expose the abdominal organs, incise the peritoneum. Next, use a fine forceps to grasp one of the uterine horns, and retract the tissue from the abdomen.
Separate the uterus from the mesometrium and fat tissue. Make the first cut between the oviduct and ovary and a second next the oviduct-uterus junction while including a small part of the uterus. Place the oviducts in the same flesh dish.
To collect zygotes under the dissection microscope, use a number five Dumont forceps to grasp the side of the uterine horn. Insert a homemade needle into the lumen of the oviduct from the infundibular end. Then, flush the oviduct with 300 to 400 microliters of HECM-9 medium.
Immediately after flushing, collect the zygotes;and wash them twice with HECM-9 in the handling dish. All zygotes should be denuded from the cumulous cells at this stage of development. Right after that, place the zygotes in an incubator until the injection to avoid exposure to light.
To prepare for injection, thaw the Cas9-sgRNA RNPs on ice. Immediately before the injection, load the injection needle with RNP solution by placing the rear end of the needle into the tube containing the solution allowing it to fill the needle by capillary action Next, fill the holder pipette with mineral oil up to approximately three millimeters of the needle bend. Place the holder in the micro injector with the tip of the holder pipette horizontal to the bottom of the dish.
Fill the needle tip with medium by suction. Then, set the injection needle at a 10 to 15 degree angle opposite to the holder. Next, identify a zygote, and use the holder needle to apply a fine suction.
Male, which is slightly bigger, and female pronuclei should preferably be within the same focal range and aligned parallel to the holder. Using the injection needle at a three o'clock position, penetrate the zona pellucida and male pronuclei. Once the pronucleus swells, withdraw the injection needle quickly to prevent the nucleus from adhering to the needle and to avoid damaging it.
Efficiency of gene targeting for STAT2 and RAG1 genes by the Cas9/sgRNA system is evaluated by live birth rate of recipient females and the number of live pups with the intended genetic modifications. Genotyping results from a PCR restriction fragment length polymorphism assay, or PCR-RFLP, of pups, show the efficiency of gene targeting. Lane one indicated biallelically targeted alleles, and lanes three and seven show monoallelically, or mosaically, targeted alleles.
This technology has paved the way for researchers to develop newer genetically engineered hamster models of human disease. After watching this video, you should have a good understanding of how to produce genetically engineered Golden Syria Hamsters by a pronuclear injection of the CRISPR/Cas9 Complex.
