Morpholino-mediated Regulation of Cell Cycle Proteins

Morpholino antisense oligos (MO; Genetools) designed to recognize the 5′UTR of cyclin B1, Cdc20 and APC3 were microinjected at a micropipette concentration of 1 mM. Cyclin B1 MO and cyclin B1 5 base pair mismatch (5bpmm) MO oocytes were released from arrest within 30 minutes of injection since we did not want to perturb prophase levels of cyclin B1, but only to prevent such excessive accumulation in prometaphase. APC3 MO and APC3 5bpmm MO injected oocytes were arrested in prophase with IBMX for 6 hours prior to release. A short Cdc20 MO incubation period (1.5-2 hours) was carried out as previously described (Reis et al., 2007 (link)). A longer Cdc20 MO incubation period required the use of cycloheximide since oocytes began to escape IBMX induced prophase arrest approximately 3 hours after injection of the Cdc20 MO. We reasoned that, without sufficient Cdc20 available to suppress prometaphase cyclin B1 levels, a premature increase in cyclin B1 drove oocytes out of prophase. Therefore, a longer Cdc20 MO incubation period included cycloheximide to prevent this. To release prophase arrest, both IBMX and cycloheximide were washed out from oocyte containing media; the use of cycloheximide in prophase arrest did not perturb subsequent MI maturation or cyclin B1 destruction profiles in control oocytes.