Late Cold Shock Gynogenesis
Late cold shock gynogenesis prevents the first mitotic cell division and therefore creates an embryo that is homozygous at all loci. Late cold shock gynogenesis is not as efficient as early cold shock since the embryos do not tolerate the procedure as well.Protocol for Diploidizing Haploid Embryos using Cold Shock Treatment-LATE (Equivalent of Late Hydrostatic Pressure) PDF
1. Make 1.0 L of 0.05X MBS + 0.1 % BSA (pH 7.50) Pre-chill several 50-mL conical tubes containing 0.05X MBS + 0.1% BSA in a slushy ice water bath (T = 1.0 to 3.0oC). Keep remaining solution at room temperature to use prior to and after cold shocking embryos to avoid any effects of concentration gradient or pH difference.
2. Euthanize male and dissect testes out. Place pair of 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 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 uJ/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 0.05X MBS + 0.1% BSA (pH 7.5 + 0.1, T= approximately room temp ~22-25oC) and start timer. Place embryos in a 22-230C water bath.
4. At 48 minutes post flood, remove media and replace with pre-chilled 0.05X MBS + 0.1% BSA (T = 1.0 to 3.0oC). Incubate embryos in a slushy ice water bath for 5 minutes.
5. Remove cold media and replace with 0.05X 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 0.05X MBS + 0.1% BSA to remove cysteine.
This protocol was kindly provided to us by Rob Grainger.