The overall goal of this procedure is to develop novel rabbit models for the study of human cardiovascular diseases. This is accomplished by first designing and synthesizing zinc finger nucleases that target the gene of interest. The second step of the procedure is to micro inject the ZFN RNAs into the cytoplasm of Pronuclear stage embryos.
The third step is to surgically transfer the embryos to a recipient rabbit. The final step is to genotype the newborns to identify transgenic animals. Overall gene targeting through ZFN is highly effective and is demonstrated here by creating an A OC three knockout rabbit.
This technique, it's the landmark in genetic engineering at it is the first to enable production of no-code rabbit. The implications of this technique extend toward therapy of many human diseases such as cardiovascular diseases. It is increasingly realized that large animal models are needed to better mimic the physiology and pathology of humans.
We first had the idea for this method when we read the article reporting successful gene targeting using significant nuclease in red in 2009. Generally, individuals knew to this method will struggle because it involves cyto plasma micro injection of construct. Though this method can provide insight into gene targeting rabbits, it could also be applied to other systems such as pigs.
Start with super ovulating, sexually matured New Zealand white rabbits using a subcutaneous injection of FSH twice a day over a period of three days, increase the dosage daily. 72 hours after the last FSH injection inject 200 IUs of HCG intravenously to induce ovulation individually made the super ovulated females immediately thereafter. At this time, a recipient animal must also be prepared.
16 to 18 hours post insemination euthanize the super ovulated donor rabbits with sodium pentobarbital. Recover the UCT pule by carefully collecting the entire uterus. Ucs and ovaries structure flush each UCT with 10 milliliters of manipulation medium by injecting the medium from the uterus end of the uct.
Collect the flushed medium at the ovary end of the UCT in a Petri dish. The recovered embryos are found in the flushed medium. Next, wash the embryos three times with manipulation medium.
Then incubate the embryos in the manipulation medium at 38.5 degrees Celsius in normal air. At 19 to 21 hours. Post insemination, select embryos using a stereo microscope.
Collect about 30 embryos that are at the pronuclear stage for each injection condition, expect 30 to 40 such embryos per super ovulated female. In advance. Obtain ZFN sets for the gene of interest from a manufacturer.
Selecting those with the best ZFN scores. In this example, the gene of interest is A OC three. To prepare the ZFN mRNA first transcribe the ZFN messenger, RNA in vitro and polyadenylate the transcripts.
Next, purify the resulting mRNA and resus. Suspend it in RNA spree 0.1 xte E.Then measure the RNA concentration using a spectrophotometer and prepare aliquots of the mRNAs encoding ZFN pairs at four to 10 nanograms per microliter in 10 microliter vials for storage. Begin with heating the microinjection stage to 38 degrees Celsius.
To prepare the microinjection platform, first, remove the media chamber from a one well chamber Slide then at a five microliter drop of manipulation. Medium to the slide and cover it with mineral oil. Place the prepared slide on the heated microscope stage.
Next, secure a 120 micron diameter holding pipette to a microm manipulator and position the pipette into the drop of medium on the platform. Now thaw the mRNAs to be injected on ice. Load them into an injection pipette, forged from a bo silicate glass capillary tube using a micro pipette puller.
Next turn on the compressed air supply connected to the micro injector. Next, secure the injection pipette to its holder and attach that to a second microm manipulator. Position this pipette into the drop of medium as well.
Now configure the micro injector for an injection volume of one to five picoliters. To open the tip of the injection pipette. Gently tap it against the holding pipette.
Using a glass pipette transfer 30 to 50 embryos to the micro manipulation. Drop on the platform. Then at 100 x magnification.
Use the holding pipette to capture an embryo. Align the holding pipette and embryo to the injection needle now under 400 x. Advance the injection pipette through the embryo.
Be careful to avoid the nucleus. Once the plasma membrane is pierced, press the foot pedal to inject the mRNA after the injection, withdraw the needle and release the embryo. Repeat the process for all remaining embryos.
Once they're all injected, wash the embryos three times in embryo culture. Medium for 30 seconds per wash. Then incubate the embryos at 38.5 degrees Celsius with 5%carbon dioxide air for one to two hours before transferring the embryos to the synchronized recipient.
Female rabbit. To culture the embryos to the blast assist stage, the incubator must have humidified air. Select a five to 12 month old female NZW Rabbit that is in good health and that weighs four to five kilos.
Prepare her as the recipient animal by first administering gonadotropin, releasing hormone intramuscularly. 16 to 24 hours after the hormone injection, anesthetize the rabbit with ketamine and or vaporized isof fluorine. When the rabbit is fully unconscious, it will not produce pedal reflexes.
When the footpad is squeezed before the surgery, protect the eyes with ophthalmic ointment and prepare the abdomen. During the surgery, record the rabbit's vital signs. Every 15 minutes begin by making an incision of approximately 2.5 centimeters through the skin and then along the muscle layer.
Exteriorize each ovary along with the attached uterine horn, holding an ovary between thumb and forefinger. Transfer the embryos to the oviduct through a carefully inserted sterile pipette. Usually 10 to 25 embryos from every 30 injected embryos are viable and they can all be transferred to one recipient.
Complete the surgery by closing the muscle layer with a coated absorbable suture. Suture the skin with either an absorbable subcuticular suture or with simple interrupted non-absorbable skin sutures. Then keep the animal warm and monitor it until its stern.
Recumbent is attained. 16 ZFN pairs were initially designed. The targeted disruption sequence of set one is located at exon two of a OC three sets.
One, two, and three were selected and subjected to the yeast. MEL one reporter assay to determine the ZFN activities set, one had a ZFN score of 224.2%higher than those of sets two and three and much higher than the selection threshold, which is 50%of the internal positive control. Therefore, set one was selected for in vitro validation experiments set one mRNA was micro injected into 35 rabbit embryos.
The BL rate of the micro injected group appeared lower compared to those that were not micro injected, indicating a slightly impaired developmental competence. 18 embryos of the micro injected group developed to the BL stage on day five of which 16 were sequenced. The frequency four mutations at the target site was much higher than the selection threshold revealing that the set one ZFN pair was effective In total 145 embryos.
Micro injected with set one ZFN mRNA were transferred to seven pseudo pregnant recipient rabbits. Freshly flushed embryos without ZFN micro injection were transferred to recipients as the control group after 30 days of gestation. 21 kits were born in the micro injected group.
PCR sequencing identified five as positive knockouts. The indels at the targeting site included two insertions and three deletions. Bioinformatics analysis was used to identify potential ZFN off targets with seven or fewer mismatches to the set one sequence, only one possible off-target site was identified and PCR detected no events at that site in any of the founder animals.
A watch this video. You should have a good understanding of how to produce knockout rabbits through zinc. Finger nucleus microinjection.
Once mastered embryo collection, microinjection and transfer can be done in four hours if it is performed properly. While attempting this procedure, it's important to remember to, the injection material is RNA, which requires extra caution. Following this procedure, phenotyping will be performed to understand the biological rules of the targeted genes.
