TTwo years after scientists at Scotland’s Roslin Institute gave birth to Dolly the sheep, the first mammal successfully cloned from an adult cell, researchers at the University of Hawaii reported another groundbreaking first for the world of reproductive technology: they had cloned mice, not from fresh, fully functioning cells, but from freeze-dried sperm.
Nearly 25 years later, some of those same researchers have figured out how to combine the techniques, a breakthrough that could be a boon to biodiversity banking and species conservation efforts.
On Tuesday, a team led by Teruhiko Wakayama, now at Yamanashi University in Japan, showed that somatic cells, not sperm or egg cells, can be used to clone healthy mice, even after they have been freeze-dried and stored for up to nine months. . . The latest researchpublished in Nature Communications, opens up new possibilities to capture and perhaps one day reconstitute the hundreds of thousands of species that are being wiped off Earth at catastrophic rates.
“From a conservation point of view, innovation of new ways to biobank reproductively viable tissue types is a great need,” said Ben Novak, principal scientist at revive and restore, a nonprofit organization founded in 2012 to investigate how biotechnology could benefit extinct and endangered species. “So it’s really exciting to see this kind of progress.”
The usual method of storing tissues collected from animals is to submerge them in tanks of liquid nitrogen, which require regular top-ups to keep them sailing at a cool -385 degrees Fahrenheit. In contrast, lyophilized cells could be stored in small, vacuum-sealed glass vials at room temperature, a cheaper solution that is also less vulnerable to natural disasters and unforeseen supply chain issues.
Pharmaceutical companies routinely use freeze-drying to preserve protein drugs and vaccines in a dry form to ensure they can be stored for long periods of time and shipped to remote destinations without the need for a costly cold chain prone to logistical disruption. Extending this method to cell and tissue preservation would allow biologists to work even in remote parts of the world to save genetic material from the species that live there.
To carry out the freeze-drying process, Wakayama’s team collected supporting cells known as cumulus cells around the oocyte of female black-furred mice. They then immersed these cells in liquid nitrogen, before lowering the pressure until all the frozen water inside the cells sublimed into steam.
The crumbly powder that remained was not alive (the freeze-drying process killed all the cells), but it contained intact DNA.
And when Wakayama and his colleagues rehydrated the lifeless dust months later, they were able to transfer hundreds of nuclei into eggs from other mice, creating early embryos called blastocysts. The researchers then removed some of the approximately 300 cells in each blastocyst and established embryonic stem cell lines in the lab. It was the nuclei of these cells that were fused with another round of eggs collected from a strain of mice with black fur, and then transferred into the wombs of mice with white fur.
Of about 2,000 embryos transferred, 19 mice were born. Their hair was black, they had all four legs, and they looked perfectly healthy otherwise. When Wakayama’s team repeated this process with cells cultured from the tips of the tails of male and female mice, they were able to produce another 56 cloned mice.
The first mouse born, a female the researchers named Dorami after a popular Japanese cartoon robot, was among a dozen mice the researchers grew to maturity and then paired for mating. She and all the other females gave birth to their own litters of healthy, screeching pups. All males showed normal fertility and produced offspring.
While the other clones were sacrificed for further analysis of their tissues, the scientists kept Dorami close to see if the process that had created her could shorten her time on Earth. She died a month and a half before her second birthday, a fairly average life expectancy for a lab mouse.
“It was really amazing that so many offspring were born,” Wakayama told STAT by email. He attributed the success to “the accumulation of small factors” in the last two decades: discoveries of optimal recipes for protective chemicals and improved techniques for transferring genetic material made in his laboratory. But he didn’t always believe it would be possible.
After he and his colleagues published their initial results with freeze-dried sperm in 1998, other scientists applied the technique to clone other species, including rats, hamsters, rabbits, and horses. Freeze-dried mouse sperm that spent six years being blasted by radiation aboard the International Space Station still produced pups, Wakayama’s team reported last year.
But the sperm turned out to be special. Their tightly bound chromosomes are packed into supercondensed nuclei, so there’s less room for water molecules. Somatic cells, by contrast, have a looser, more liquid nuclear structure, making them more prone to DNA damage as water is removed. For more than two decades, efforts to freeze-dry anything but sperm failed.
Still, there was a good reason to keep trying. As habitat loss, climate change, and other human activities drove more species to extinction, conservation groups began pushing to preserve the genetic material of the world’s creatures in biobanks, or “frozen zoos.”
Sperm and eggs would be ideal, but obtaining eggs from any animal, let alone a wild animal, is no easy task. It requires an ultrasound-guided needle and specially trained technicians to use. (Oocytes can also be collected from freshly dead animals, but most species don’t expire near an assisted reproductive technology lab.) Sperm is easier to collect, but it is not always possible, especially with old and infertile animals. And saving the sperm only saves the males; Without eggs, half of the genetic diversity of a population is lost.
Skin cells, on the other hand, can be easily and reliably harvested without causing harm. And they carry both sets of chromosomes coiled up inside their nuclei.
“Being able to open and salvage somatic tissues from both males and females is really an essential need for genetic management,” said Novak, who was not involved in the new research. The study shows that it is possible with mice, something Novak called “an incredible feat.” But it is only a first step. The method was not very efficient, and unlike freeze-dried sperm, freeze-dried somatic cells still had to be kept in a -22 F cooler, which requires electricity. In some regions of the world, it’s easier to keep liquid nitrogen tanks full than it is to keep the power on.
The laboratory mouse is also among the animals whose biology is best understood. With other less studied species, knowledge about how best to preserve somatic cells and produce new animals from them falls precipitously.
Still, knowing something like this is possible is a big deal for people like Novak. “Being able to save things now is more important than having the technology to clone or reproduce something from that material,” he said. “As long as we have intact nuclei or preserved living cells in some way, eventually someone will come along and innovate the technology to use them.”
The Wakayama group hopes to give conservation efforts even more options for collecting genetic material in the future. He is working on the recovery of long-frozen animal nuclei and cells shed in urine and faeces.