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Updated: Jun 3, 2026
Mouse Embryonic Development in a Serum-free Whole Embryo Culture System
Published on: March 1, 2014
Preparation of culture media for fertilized one-cell mouse eggs
1Neuropeptide Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Republic of Singapore.
Abstract:
Two types of culture media 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 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.
Insights
Two specialized culture media, M16 and M2, are essential for in vitro manipulations in creating transgenic mice. M16 is for incubator use, while M2 supports brief periods outside the incubator.
Area of Science:
- * Reproductive biology and developmental science.
- * Genetically modified organism development.
Background:
- * Transgenic mouse generation requires precise in vitro culture conditions.
- * Maintaining oocyte viability during manipulation is critical.
Purpose of the Study:
- * To describe the distinct roles and compositions of two essential culture media for transgenic mouse production.
- * To highlight the limitations of each medium for specific experimental procedures.
Main Methods:
- * Description of M16 culture medium for incubator use.
- * Description of M2 culture medium for ex-incubator manipulations.
- * Comparison of buffering systems (bicarbonate vs. HEPES) and their impact on pH stability.
Main Results:
- * M16 medium, buffered solely by bicarbonate, is suitable for incubator use but sensitive to pH changes outside.
- * M2 medium, supplemented with HEPES buffer, allows for short-term (≤30 minutes) oocyte maintenance outside a CO₂ incubator.
- * Oocytes require specific pH conditions for survival during in vitro procedures.
Conclusions:
- * The choice of culture medium (M16 or M2) is dictated by the experimental procedure's location (in or out of the incubator).
- * M2 provides a temporary solution for oocyte handling outside the incubator, but prolonged exposure is detrimental.
- * Proper medium selection is crucial for successful transgenic mouse generation.
