Early Cold Shock Gynogenesis
Early cold shock gynogenesis prevents extrusion of the polar body and is therefore effective for making haploid embryos diploid and making loci near the centromere homozygous. Early cold shock gynogenesis is more efficient than late cold shock gynogenesis, although only a portion of the genome is made homozygous.Protocol for Diploidizing Haploid Embryos using Cold Shock Treatment (Equivalent of Early Hydrostatic Pressure) PDF
1. Make 1.0 L of 0.05 X MBS + 0.1 % BSA (pH 7.50). Pre-chill several 50-mL conical tubes containing 0.05 X MBS + 0.1% BSA in a slushy ice water bath (T = 1.0 to 3.0 oC). Keep remaining solution at room temperature to use prior to and after embryo cold shock embryos to avoid any effects of concentration gradient or pH difference.
2. Euthanize male and dissect testes. Place both testes in an Eppendorf tube containing 100 uL 1X MBS + 0.1% BSA. Homogenize testes using a sterile micro pestle and rinse the pestle with 500 uL of 1X MBS + 0.1% BSA into the Eppendorf containing the sperm slurry. Transfer the total volume of sperm slurry into a 15 mL conical tube containing 1 mL of 1X MBS + 0.1% BSA. Allow slurry to settle for approximately a minute. Aliquot 250-300 uL of the supernatant containing sperm into petri dishes for in vitro fertilization. Irradiate sperm. Using our Fisher Biotech XL-1000 UV-Crosslinker, sperm is irradiated at 600 x 100 µJ/cm2.
3. Collect dry eggs from female and aliquot 250-1000 eggs per dish of irradiated sperm. Swirl the dish gently and wait 5 minutes. Flood the embryos with a small amount of the remaining 0.05 X MBS + 0.1% BSA (pH 7.5 + 0.1, T= approximately room temp ~22-25oC). Let sit at room temperature for 5 minutes.
4. Remove media and replace with pre-chilled 0.05 X MBS + 0.1% BSA (T = 1.0 to 3.0oC). Incubate embryos in a slushy ice water bath for 10 minutes.
5. Remove cold media and replace with 0.05 X MBS + 0.1% BSA (T = ~22-25oC). Allow to develop to 4-cell stage and dejelly with 2% cysteine (pH 7.9 + 0.1). Rinse embryos a minimum of 5 times with the remaining 0.05 X MBS + 0.1% BSA to remove cysteine.
This protocol was kindly provided to us by Rob Grainger.