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Cryopreservation - vitrification method

This, the first such review, is a synopsis of an article published in early 1995 in the journal Transgenic Research by a Japanese group, Shyoso Ogawa et al. Although not a particularly recent article, I would contend that it demonstrates a technique shown in other labs while suggesting a useful methodology that may prove useful in many transgenic laboratories.

Japanese labs have long been at the forefront of cryopreservation technology, and have a strong commitment to research in this area. This paper further strengthens the technique of embryo vitrification as a successful means of cryopreservation, while suggesting that the viability of these embryos post-thaw undergoing DNA microinjection, although reduced over non-cryopreserved embryos, is still a plausible alternative.

Title: Production of trangenic mice by microinjection of DNA into vitrified pronucleate stage eggs

Authors: M. Tada; M. Sato; K.Kasai and S. Ogawa

Reference: Transgenic Research, Vol. 4, pp208-213 (1995)

Abbreviated methodology

Embryo isolation / fertilization
1. Female mice (B6C3F1 hybrid) were given PMSG / HCG treatment to induce superovulation, and unfertilized eggs removed 14-16 hrs post-HCG.
2. In drops containing 20-30 oocytes apiece, a sperm suspension was introduced for the purpose of IVF. By 6 hrs almost all had been fertilized.

Vitrification / thawing

3. Using a DPS mixture (DMSO/propylene glycol/sucrose) as a cryoprotectant, the now fertilized eggs were placed in Nunc cryotubes and plunged into liquid nitrogen, and stored for 3-6 months.
4. Thawing was achieved by immersion in a 40 degree waterbath and supplementation of PB1 media with a reduced sucrose concentration. After washing, the eggs were incubated 1-2 hrs prior to microinjection.

Microinjection

5. Microinjection was performed by standard methodology. Microinjected eggs were cultured overnight in an incubator. Only resulting 2-cell embryos were implanted into ICR pseudopregnant recipients

Results

There appears to be a reduction in the numbers of eggs developing to 2-cell by about 25%, and this is held up by the numbers of transgenic live young.

This paper, which quotes comparable rates for these studies with controlled-rate freezing methodology, suggests the following:
1. Vitrification is a viable and equal alternative to controlled-rate freezing technology, and in many cases is more cost effective for most labs.
2. Vitrification may prove to be a useful tool in a production setting, where many embryos can be banked for use later.
3. Use of this technique allows for microinjection to occur independent of time of day, room light cycle, available technical help or other variables.


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