The overall goal of this procedure is to rapidly generate gene deficient mice with the help of designer tail nucleases or talons. This is accomplished by first designing and assembling DNA constructs encoding specific talon pairs, which serve as templates for in vitro transcription to generate Talon mRNAs. The next steps of the procedure are to isolate fertilized mouse cytes, and then micro inject them with the T.The final step is to surgically transfer the injected cytes into the pseudo pregnant foster mothers.
The resulting F zero progeny frequently show site-specific deletions of genomic sequences that often lead to the loss of target gene function. Although the method presented here can provide insight into gene function in mice, the basic approach can also be adapted to other model organisms such as rats and fish. First, access the TAL effector nucleotide targeter website on the site.
Choose the TN targeter program and paste in the sequence of the target gene. For this protocol. Use the P-C-A-G-T seven TALEN expression constructs and the golden gate Talen and T effector kit that are available on ad gene.
If other constructs or assembly kits are used for Talen assembly settings might be different for the optimal spacer length and numbers of tail RVs, choose the Miller Etal 2011 option under the use a preset architecture setting. Then select NN under the G substitute tab. The program generates a text file with potential target sites generated from this list.
Select the Talen pair or pairs required and proceed with the Golden gate assembly. This protocol works with most commonly used laboratory mouse strains, including C 57 BL six J and BDF one super ovulate 10 donor females at four weeks of age using an intraperitoneal injection of five units of pregnant mare serum gonadotropin. 48 hours later give the females five units of human chorionic gonadotropin also by intraperitoneal injection immediately after the human chorionic gonadotropin injection mate, the females one-to-one with breeding age males on the same day, prepare the pseudo pregnant foster mothers.Others.
The next morning, sacrifice the females and recover the fertilized oocytes by dissecting out the ucts. Then release the oocyte clutches into an organ culture dish containing a milliliter of M two medium. Remove all of the cumulus cells by adding 10 microliters of 0.1%bovine hyaluronidase solution to a dish containing cyte clutches in M two medium.
Then use the mouth pipette to wash the embryos through two or three changes of M two medium to remove the hyaluronidase. Continue the hyaluronidase treatment for embryos that have not yet dissociated from cumulus cells. The isolated embryos can be stored in M 16, medium at 37 degrees Celsius and 5%carbon dioxide until they're micro injected.
Prepare 100 microliters of injection solution containing 10 nanograms per microliter of each tail. Nuclease, mRNA, pair, and centrifuge for 30 minutes at 30, 000 G to pellet. Any particles that may clog the injection needles.
Then keep the mix on ice. Plan on injecting the oocytes at their pronuclear stage in the early afternoon. Submerge approximately 100 embryos in M two medium under a layer of mineral oil and work on an inverted microscope with no marsy DIC optics.
Under 20 x magnification, select cytes with distinct pronuclei. Sorting of oocytes can be done using an empty injection capillary load the injection capillary just before it is used with one to two microliters of the injection solution. Then attach it to the microinjection head.
Then aspirate a selected cyte with a holding capillary and make a shallow injection of the nuclease mRNA into the cytoplasm. Avoid contact with the pronuclei. The injection volume should be kept low.
At the first sign of cytoplasmic distension, withdraw the needle. Normally, about 80 to 90%of the embryos should survive without immediate. To increase the amount of injected mRNA, make the solution more concentrated following microinjection of the available cytes.
Transfer them into M 16 media using a mouth pipette. Incubate the embryos for an hour at 37 degrees Celsius and 5%carbon dioxide. Then transfer the ones that survive into pseudo pregnant foster mothers.
The day before the microinjection induce pseudo pregnancy in three to six month old CD one female mice by mating them to VAs sectorized males. The next morning select females with a clear atory plug to use as embryo recipients. Unused females will revert from their pseudo pregnant state in three weeks and can be reused afterwards.
Position an anesthetized foster mother female over a warm surface on its abdomen. Then disinfect the area of the incision by spraying it with 70%ethanol. Comb the wet hair away from the planned incision site.
Now cut open the peritoneal cavity with scissors and externalize the uterine horn. To do this. Pull on the fat pad attached to the ovary and immobilize reproductive organs with a bulldog clamp.
Also, be sure to keep the organs moist with warmed 0.9%sodium chloride solution. At this point, sort 12 to 20 viable embryos and load them into a thin glass capillary. Using the mouth pipette first aspirate a small air bubble, then aspirate up the embryos using fine forceps.
Gently grasp the UCT just upstream of the amp and create a small hole in the UCT wall using a 30 gauge needle. Insert the loaded capillary into the hole and gently blow the embryos out into the amp until you see that the air bubble has been displaced from the capillary. Then remove the capillary.
Immediately replace the reproductive organs back in the body cavity and close the peritoneal cavity with a single suture. Next, close the skin using one or two nine millimeter wound clips. Return the animal to its home cage and supervise her until the anesthesia wears off.
Then house the females in stable social groups of two to three mice to minimize stress for the first three postoperative days, be sure to provide an analgesic like DEF Falcon in the drinking water for every locust in the mouse genome that is targeted with designer nucleases. A PCR based assay is designed to identify founder animals that carry a desired mutated allele. In the first example, founders originating from the microinjection of a tail nuclease pair.
Targeting the coating region of the mouse prion protein gene were analyzed by T seven endonuclease digestion of a PCR product. The T seven Endonuclease digestion assay relies on the formation of hetero duplexes between DNA strands of PCR products generated from mutated and wild type alleles. Talon induced mutagenesis within the targeted genomic region of single founders was revealed by the appearance of 250 and 110 base paired long digestion products in addition to the full length PCR product.
Alternatively, PCR products can be subjected to a restriction digest if a suitable diagnostic restriction site is located within the spacer region of the designer nuclease pair. In the second example, ZFN targets an XPO one restriction site located within the intro, one of the mouse Rosa 26 locus. Here undigested bands indicate the presence of mutations.
The asterisks mark, the absence of any digested products strongly suggesting a founder carrying mutations in both alleles of the targeted gene. Cloning and sequencing of these undigested PCR products reveals that founder mice can harbor two or more distinct modifications of the targeted allele. The absence of wild type sequences indicates the complete ablation of the target gene.
After watching this video, you should have a good understanding of tail nuclease design, assembly of tail, nuclease constructs, and how to use these to generate mutant mice by direct oside microinjection. This method can be adapted to work with other classes of designer nucleases, such as zinc, finger nucleis, or CAS nine crispr. It can also facilitate genome modification by homologous recombination through simultaneous co injection of the nuclease and the appropriate gene targeting template.
