New method advances long-term storage of genetic material

As species continue to face mass extinction, preserving genetic material through judicious biobanks to enable cloning is essential to promote species survival and maintain biodiversity.

Embryos and germ cells are usually stored at ultra-low temperatures in liquid nitrogen. But storing reproductive cells in liquid nitrogen is tricky. Not only does this require considerable skill and maintenance costs, but it also requires regular monitoring, and biomaterials stored in liquid nitrogen can be easily destroyed during power outages and other disasters. Therefore, it is crucial to develop methods where genetic material can be stored long-term with minimal resources that allows the cloning of viable and fertile offspring during prolonged storage.

Scientists at Yamanashi University in Kofu, Japan, have developed a new method that uses freeze-dried somatic cells – cells other than reproductive cells – to clone mice. The results were published in a journal article Nature Communication titled, “Healthy Cloned Offspring Derived from Freeze-Dried Somatic Cells” on July 5, 2022. Teruhiko Wakayama, PhD, Professor in the Department of Environmental Sciences at Yamanashi University is the lead author of the study. The authors say the new method could be used to store genetic material from any animal safely and inexpensively.

In an earlier study, Wakayama’s team had developed a technique for freeze-drying mouse sperm. Although the researchers were unable to recover healthy, functioning sperm after the freeze-drying process, they were able to recover DNA from sperm which they injected into oocytes to clone mouse offspring. They then demonstrated that the technique worked in other species, including rats, hamsters, rabbits and horses. This increased storage stability and security.

Researchers have observed that freeze-dried mouse sperm are highly resistant to environmental fluctuations. Wakayama’s team successfully obtained healthy mouse offspring from air-dried sperm stored in a desk drawer for over a year and on the International Space Station for over 5 years. This convinced Wakayama and his team that freeze-drying is the best and safest way to store genetic material for long periods at low cost in any location.

However, until now, whole animals have only been cloned from freeze-dried mature sperm. The collection of functional sperm, especially from infertile males, as well as the collection of female eggs from fertilized ovaries or embryos pose significant challenges for the biobank.

Since Wakayama reported the cloning of whole animals from freeze-dried sperm DNA, frogs and sheep have been successfully cloned from somatic cells, indicating that gamete storage is not essential in as a genetic resource. Additionally, somatic cells can be easily collected from anywhere in the body, including bodily waste and after death.

In the current study, Wakayama’s team generates healthy cloned mouse offspring from freeze-dried somatic cell nuclei through an adapted nuclear transfer procedure. They used fibroblast cells from the tail tips of mice. “Our data reveal that although some DNA abnormalities are observed during the process, lyophilized somatic cell nuclei can be used to generate blastocysts by nuclear transfer, and embryonic stem cell lines derived from these blastocysts produce donor nuclei capable of producing healthy and fertile blastocysts. cloned mice,” the authors noted.

The researchers freeze-dried the somatic cells for up to nine months at -30°C, using trehalose as a cryoprotectant or epigallocatechin as an antioxidant. When they attempted to revive the cells with rehydration and stain them with propidium iodide, they observed that the cell membranes were damaged and the cells died.

Nevertheless, the authors used DNA from these cells as donor material for cloning to generate healthy female and male offspring, with a success rate of 0.2-5.4%. They adapted the somatic cell nuclear transfer procedure to generate stable embryos (blastocysts) and embryonic stem cell lines.

“After nuclear transfer, we produced cloned blastocysts from freeze-dried somatic cells and established embryonic stem cell lines by nuclear transfer,” the authors noted.

Additionally, the investigators selected nine cloned female and three male mice and allowed them to mate. All females gave birth to litters, indicating that fertility is retained in cloned animals.

Comet tests on the freeze-dried cells revealed that the DNA of these cells suffered more damage than that observed after storage in liquid nitrogen. However, according to the authors, the resulting cloning success rate indicates that the new method may provide a viable alternative despite DNA damage, as it provides a cost-effective and long-term solution.

“We show that freeze-dried somatic cells can produce healthy and fertile clones, suggesting that this technique may be important for the establishment of cheaper and safer liquid nitrogen-free biobanking alternatives,” the authors concluded. .

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