This is an alternate and easy to use method for delivering genes of interest to fertilized mouse eggs via laser perforation of the zona pellucida and lentiviral gene delivery. Laser-assisted perforation of the mouse donor procedure may also be applicable to the fertilized eggs of other species for facilitating the entry of other types of viruses or transfection reagents. The main advantage of this technique is that it requires no specialized skills for the micromanipulation and micro injection of the fertilized mouse eggs.
Demonstrating fertilized mouse egg isolation procedure will be Page Myers, a research scientist from the Comparative Medicine branch at the NIEHS. 16 to 24 hours post mating, isolate the ovaries and ovaducts from female mice with vaginal plugs, according to standard protocols. When all of the tissues have been collected, tear the ampulla of each ovary apart, and release the fertilized eggs, surrounded by cumulous cells, and transfer the eggs in cumulous cells to a 35 millimeter dish containing one x hyaluronidase in fresh M2 medium, for a three to five minute incubation at room temperature.
Pipette the eggs a few times to release the cumulous cells, and transfer the eggs to a new 35 millimeter dish containing M2 medium, with pipetting to wash off the enzyme. Next, transfer the washed eggs into a 35 millimeter dish containing potassium simplex optimized medium, or KSOM, and pipette to wash off the remaining M2 medium and hyaluronidase. Then, transfer the eggs to 35 millimeter tissue-culture treated plates seeded with 50 microliters of KSOM and two milliliters of dimethylpolysiloxane for a two hour equilibration at 37 degrees Celsius, 5%carbon dioxide and oxygen, and 90%nitrogen.
While the eggs are in the incubator, set up and calibrate the XYClone laser, according to the manufacturer's recommendations. When the eggs are ready, place the first drop plate onto the laser microscope stage, and manually focus the zona pellucida. After confirming that the laser LED light is visible through the objective, move the microscope stage to target the zona with the LED laser, and use the computer software to set the XYClone laser to 250 microseconds.
Adjust the LED light size to the appropriate dimensions, and perforate the zona pellucida of each egg three times with the laser to produce three 10-micrometer holes. It is critical to perform the perforation quickly, but carefully, as the fertilized eggs can not keep outside of the incubator for longer than 15 minutes. Then, return the perforated eggs to the incubator for another two hours.
At the end of the second incubation, treat the 50 microliter KSOM drop with two microliters of concentrated lentivirus without mixing. And return the eggs to the incubator for four days. When the eggs have developed into blastocysts, use a non-surgical embryo transfer device, to deposit approximately 10-15 embryos in an about two microliters of KSOM.
17 days after the embryo transfer, allow the pregnant mouse to give birth naturally or collect the neonates by cesarean section as necessary. Then, collect tissue from the pups for genotyping, to determine the rate of transgenesis. Isolated and transduced mouse-fertilized egg development can be checked under the microscope daily.
60 to 70%of healthy, untreated embryos develop into blastocysts within three to four days. While about 47%of laser-perforated, transduced embryos develop into blastocysts, 85%of which express the tranduced gene of interest. Aiming the laser to thin the zona, instead of creating a hole, allows the developing embryos to benefit from an intact, encapsulating zona pellucida while remaining permissive to lentiviral transduction.
Transduction with a recombinant lentivirus, expressing copepod GFP from an elongation factor 1A promoter induces multiple lentiviral integrations, and a high expression of GFP under a blue LED lamp. While attempting this procedure, it is important to remember that, while the ability of lentiviruses to integrate into the host genome makes them a powerful tool for stable gene delivery, but random integration may also introduce insertional mutations. This technique could enable researchers in the field of genetics to rapidly develop transgenic animal models for the study of disease for in vivo protein production, or for functional gene studies.
Following this procedure, other methods like immunohistochemistry can be performed on isolated tissue samples from transduced pups to answer additional questions about the location and the extent of gene expression in various tissues of interest. Don't forget that working with lentiviruses can be extremely hazardous. Therefore please follow your institutional guidelines for working with lentiviruses.
Wear personal protective clothes and decontaminate all waste prior to disposal.
