Acquisition and culture of mouse embryos prior to implantation

Summary

Mouse development begins with the formation of a fertilized egg, and the early development of its embryogenesis with be much slower than that of other model organisms. The mouse embryo is slowly oviposited at the 2-cell stage without any significant increase in size and moves down the oviduct toward the uterus until implantation in the uterus 4.5 days after fertilization. During this process, the fertilized egg undergoes mulberry formation (compartion) to become a solid ball of cells, the mulberry embryo (morula). The outer part of the morula forms the trophoblastic ectoderm (tmphectoderm), which will later differentiate into embryonic accessory structures such as the placenta. Inside, the inner cell mass (ICM) is formed, and part of the ICM develops into the fetus. At 5-10 days after fertilization, three basic germ layers: ectoderm, mesoderm, and terminal endoderm (distinct from the primitive endoderm) are produced by proto-gut action, and the basic body structures and organ primordia of the future mouse are established.

Operation method

Acquisition and culture of mouse embryos prior to implantation

Materials and Instruments

Equipment:
① Disposable sterile plastic container
② egg transfer tube
③ Incubator
Reagents:
①Materials: mice
②Embryo culture solution
③paraffin
④hCG
⑤ Antibiotics

Move

The basic process of micro fertilization technique can be divided into the following steps:

I. Preparation of mouse embryo culture before implantation 1. When preparing the fluid, it is best to use disposable sterile plastic containers and egg transfer tubes. Embryo culture fluid should generally not be kept for more than 1 week (maximum 2 weeks). Fresh culture fluid should be prepared within 1 or 2 weeks.

2. Culture drops for culture should be made 4 to 6 hours before culture or 1 day in advance. Covering with liquid paraffin helps stabilize the culture solution and reduces evaporation (which can lead to an increase in osmotic pressure), as well as temperature and pH changes caused by manipulation outside the incubator. Generally 20-50 μl culture drops are used.

3. For experiments involving prolonged manipulation outside the incubator, or for in vitro fertilization, it is recommended to maintain the microculture drop at 37 °C. The culture solution used for collection, manipulation and culture of embryos contains antibiotics and is filtered for sterilization.

Embryo collection 1. Mice are decapitated and the ovaries are isolated according to humane requirements and methods.

2. Hoppers can be collected a few hours before microinjection. When mice are kept in a light-dark cycle, it is convenient to collect conidia before noon. In the case of superovulation, 21 to 25 hours after hCG injection, syncytia are collected when a vaginal plug (0.5 dpc) is detected early in the morning of day 2 after mating. Unfertilized, oocyte-coated eggs used in in vitro fertilization can be collected in the same manner.

3. By the time embryos at the 2- to 8-cell stage have been present in the oviduct for 20-60 h, the embryos have been completely depopulated of the oocyte mound cells and can be flushed out of the oviduct with a small amount of M2 culture fluid.

After superovulation of donor female mice, 2-cell stage embryos were collected 45-48 h after hCG injection (1.5 dpc); 8-cell to densified mulberry embryos were collected 67-77 h after hCG injection (2.5 dpe).

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