Preparation of Culture Media for Fertilized One-Cell Mouse Eggs

Two types of culture media (1 ) are required for the in vitro manipulations involved in the process of making transgenic mice. (1) M16: This is used for maintenance of eggs in microdrop cultures (see Chapter 17 ) in a 37�C incubator gassed with 5% CO₂ . Since this medium is buffered with bicarbonate alone and the eggs are extremely susceptible to pH changes, it is not suitable for maintaining eggs outside the incubator. (2) M2: This is used for maintaining eggs outside the incubator during prolonged manipulations, such as collection or microinjection. M2 has essentially the same components as M16, although the bicarbonate is partially replaced with HEPES buffer to facilitate survival outside the CO₂ incubator. However, the eggs should not be maintained in M2 for periods longer than 30 mins.