Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

Our protocol facilitates the preparation of genetically modified mice easily, efficiently, and rapidly from single cell mouse embryos. Our protocol combine the use of freeze-thawed one-cell stage embryos and electroporation allowing the rapid generation of genetically modified mice including the mutant mice at high efficiency and low mosaic rates. For in vitro fertilization, begin by super ovulating four or eight-week-old female C57BL/6J mice via intraperitoneal injection of 7.5 international units of pregnant mare serum gonadotropin followed by intraperitoneal injection of 7.5 international units of human chorionic gonadotropin 48 hours later.

Next, remove cauda epididymis from sexually mature three to five-month-old male C57BL/6J mice and use a dissecting needle to extract clots of spermatozoa. Then incubate the clots in a 200 microliter drop of HTF at 37 degrees Celsius and 5%carbon dioxide for 1.5 hours for capacitation. 16 to 18 hours after human chorionic gonadotropin injection, collect cumulus oocyte complexes from the oviducts and incubate the complexes with 200 microliters of HTF covered with liquid paraffin for a no more than two-hour incubation in the cell culture incubator.

At the end of the incubation, add one to five microliters of sperm suspension from the boundary of the incubation medium to the 200 microliter drop of cumulus oocyte complexes and place the co-culture in the cell culture incubator for three hours. At the end of the incubation, wash the oocytes with potassium supplemented simplex optimization medium three times to remove any remaining sperm and cumulus cells and use an inverted microscope to check the pronuclear formation and grade of the one cell embryos. For one cell embryo cryopreservation, transfer the embryos into a fresh 200 microliter drop of HTF supplemented with 20%fetal bovine serum for a 10-minute incubation in the cell culture incubator.

At the end of the incubation, transfer 20 to 100 embryos to 50 microliters of one molar dimethyl sulfoxide solution and transfer up to 100 embryos in five microliters of solution to the bottom of a cryotube. Cool the embryos for five minutes at zero degrees Celsius before adding 45 microliters of DAP213 solution down the side of the tube. After capping, keep the cells for an additional five minutes at zero degrees Celsius before quickly storing the tube in liquid nitrogen.

Prepare one microgram per microliter of CRISPR RNA and one microgram per microliter of tracrRNA in reduced serum minimal essential medium. Add six microliters of each RNA solution to 42 microliters of nuclease-free buffer and place the mixture into a dry heater for three minutes at 95 degrees Celsius. At the end of the incubation, cool the mixture at room temperature for five minutes and prepare one microgram per microliter of HiFi Cas9 protein with reduced serum minimal essential medium.

Add six microliters of the diluted HiFi Cas9 solution to the RNA solution and transfer up to 100 thawed embryos into 25 microliters of the resulting ribonucleoprotein complex solution in a one whole glass slide. Then incubate embryos for 10 minutes at 37 degrees Celsius. For embryo electroporation, at the end of the incubation, set the electroporator parameters and load the slide onto the electroporator.

Electroporation should be performed within one hour of embryo thawing to ensure that genome editing occurs before the first replication of DNA and to prevent mosaicism. After the electroporation, wash the embryos three times with modified Krebs-Ringer bicarbonate buffer two medium in two washes with a drop of potassium supplemented simplex optimization medium covered with liquid paraffin. Then incubate the embryos in fresh potassium supplemented simplex optimization medium in the cell culture incubator overnight.

Using this modified cryopreservation method for one-cell embryos improves the developmental rate of the freeze-thawed embryos to the two-cell stage. Using this method, all of the generated mice in this representative experiment were albino with only one mosaic mouse bearing a coat with white and black patches. Here a representative section of a chromatogram from an albino mouse containing the M1 allele can be observed.

As illustrated, the protocol can generate knockout and knock-in mice with high efficiency and low mosaic rates. Indeed, the F1 offspring of homozygous F0 offspring are heterozygous containing one mutant and one wild type allele with no disparate mutations. Remember that the fertilized egg is fragile at one-cell stage.

The addition of fetal bovine serum reinforces the embryo. However, it must be handled carefully at each step. This method can contribute to the analysis of gene function and research in human diseases by facilitating a faster and more efficient production of genetically modified mice.