06 July 2022

Drying instead of deep freezing

Biotechnologists have cloned a mouse from lyophilized somatic cells

Anastasia Lyashenko, N+1

The results of the experiment are published in Nature Communications (Wakayama et al., Healthy cloned offspring derived from freeze-dried somatic cells).

Conservation of genetic resources for further cloning is important for maintaining biodiversity. Previously, scientists have successfully cloned several animal species using the nuclear transfer method, in which the nuclei of somatic cells (cells of the body that are not involved in reproduction) are transferred to an egg cell without its own nucleus. This method also turned out to clone the deceased Przewalski's horse, and in the future it is planned to extend the practice to other endangered species of animals. However, the storage of genetic material is costly, so researchers are looking for new methods of their maintenance and expansion of the bank of genetic material of species.

Japanese biotechnologists led by researcher Sayaka Wakayama from Yamanashi University decided to test the possibility of cloning using lyophilized somatic cells. The researchers used fibroblasts (connective tissue cells) and cumulus cells (cells surrounding the oocyte) mice that were lyophilized with trehalose or epigallocatechin. Both types of cells were kept in ampoules at a temperature of -30 for no more than nine months.


Stages of the cloning process

The cloning process itself took place in nine stages. Before the nuclear transfer procedure, biotechnologists rehydrated lyophilized cells. The analysis showed that the membrane of all cells was damaged. Next, they injected the nuclei of lyophilized cells into nuclear-free eggs and activated them. After six hours, all the embryos formed a pseudo-pronucleus, the DNA in the cells was damaged. The authors cultured the cells for 96 hours to study their ability to develop into blastocysts. Introducing them into the mouse uterus would not have given high success due to severe damage, so the researchers went the other way. After removing the shiny shell, they extracted embryonic stem cells and placed them in a multipath tablet with embryonic fibroblasts until full maturation, with a success rate of 10-18 percent. After that, they added demecolcin to the medium to stimulate metaphase and two hours later transferred the cells to a slide. Almost all cells had a normal structure and karyotype.

The cloned embryos that reached the two-cell stage were injected into the ovocytes of pseudo-pregnant female mice previously mated with vasectomized males, and then from five to eight of the resulting embryos were transferred to the horn of the mouse uterus. On day 19.5 after mating, the mice gave offspring by Caesarean section.


The first cloned mouse from lyophilized cumulus cells. She lived 676 days.

The analysis did not show any obvious abnormalities in the reproductive organs of the cloned mice, and they were able to give offspring. The overall success rate in cloning using lyophilized somatic cells was 0.02 percent, which is lower than that of the first cloned animal, Dolly the sheep (0.4 percent).

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