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At San Diego’s Frozen Zoo, a chance for animal immortality

By Amber Dance, Knowable Magazine

The last male northern white rhinoceros — his name was Sudan — died in March, leaving only two members of the subspecies behind: his daughter and granddaughter.

In the past, those stark facts would have spelled the end. But researchers at the San Diego Zoo’s Institute for Conservation Research — also known as the Frozen Zoo — are working to give northern white rhinos a second chance. Since 1975, the institute has been collecting tissues from creatures, some endangered and some not, then growing the cells in the lab and preserving them at a chilly 321 degrees below zero Fahrenheit.

Zoos already use reproductive technologies such as in vitro fertilization for animals like gorillas, and artificial insemination for pandas. (Elsewhere, scientists are considering the merits of resurrecting extinct species such as the woolly mammoth and the passenger pigeon, though they’d have to use ancient DNA for that.) The Frozen Zoo has used its preserved sperm to create pheasant chicks, for example, and has gone as far as making embryos of cheetahs and fertilizing the eggs of southern white rhinoceroses.

Now its zookeepers hope that their dozen northern white rhino samples will become parents to a new generation in a different way: using stem-cell technology to turn preserved white rhino skin tissue into eggs and sperm.

The institute’s research goes beyond baby-making. Scientists there are working on methods to genetically identify meat from primates and duiker antelopes that have been illegally hunted. And in the future, they might use its collection to restore genetic diversity to endangered black-footed ferrets.

Broadening such efforts will take a global network of frozen zoos, write Oliver Ryder, the institute’s director of conservation genetics,  and coauthor Manabu Onuma in the Annual Review of Animal Biosciences.

Ryder, who has been at the institute since it started, spoke with Knowable about the past, present and future of the Frozen Zoo. This conversation has been edited for length and clarity.

How did the Frozen Zoo begin?

We like to say the Frozen Zoo started when Dr. Kurt Benirschke came to the San Diego Zoo in 1975. Dr. Benirschke, who died in 2018, was interested in fertility and chromosomes. The technology to freeze cells and thaw them with restored function was still fairly new — it was first done with sperm in 1949 — but it opened up all kinds of opportunities. Having cell cultures to freeze, revive and grow more cells from, to look at chromosomes, was a really significant advance. Other scientists were doing this, but mostly in human medical research.

Dr. Benirschke was particularly interested in describing the chromosomes of diverse species. We took every opportunity that came up to collect a new species. There was a confidence that cells being banked would find uses far beyond what could be envisioned at the time.

You also joined the Frozen Zoo, then called the Center for Reproduction of Endangered Species, in 1975. Do you remember the first animal you ever preserved cells from?

It was a Chinese muntjac, a barking deer. There was one that had a fracture and was repaired by orthopedic surgeons. They had to catch it and anesthetize it to change the cast, so I was able to come and do a skin biopsy.

The barking deer are interesting. There are both Chinese and Indian muntjacs, and the average person would be challenged to identify much difference between the two. Yet while the Chinese muntjac has 46 chromosomes, the Indian muntjac has the lowest known chromosome number in mammals: six in females, seven in males.


Those of us interested in evolution, in how chromosomes evolved and how species are formed, were challenged by this “scandal,” as it was called. We now know that the chromosomes fused together in the ancestor of the Indian muntjac. What triggered that fusion isn’t known, but it must have taken place. A hybrid between the two was made in China; it had three big chromosomes and 23 little ones. It lived, but it couldn’t reproduce.

Another time, in 1980, I remember the pathologist telling me, “We’ve got a really interesting and rare animal in the necropsy [animal autopsy] room. You should get a sample.” And it was a northern white rhinoceros. That sample provided the material, years later, to do the first genetics study comparing northern and southern white rhinoceroses.

Now fast-forward to the present, and we have an ambitious genetic rescue project to prevent the extinction of the northern white rhinoceros, and it depends on the 12 northern white rhino cultures that we’ve banked over the years.

How can you use those cells to save the northern white rhino?

From those banked skin samples, we have frozen cells called fibroblasts. We thaw those cultures, and turn them into what are called induced pluripotent stem cells. These can, theoretically, turn into nearly any kind of cell in the body: They can make beating heart cells, they can make nerve cells.

Our eventual goal is to turn the stem cells into eggs and sperm, so that we can make embryos. In other words, those fibroblasts will be able to have babies.

This has been done in lab mice, but no other species yet. So far, we’ve got induced pluripotent stem cells from northern white rhinos. Now we’re refining methods to confirm they are pluripotent. There’s another team that’s working with a herd of female southern white rhinos that we plan to use as surrogates. We’re at least a decade away from a baby northern white rhino, but we’re making progress.

If you would have asked me in 1985, can you take a cell from the Frozen Zoo and turn it into an animal, I would have said no. Now we’re planning to do it in the lab here.

What else are you doing with samples from the Frozen Zoo?

We are an excellent source for genome projects. We have contributed samples to something like 140 whole-genome sequencing projects.

We’re collaborating with the Broad Institute in Cambridge, Massachusetts, on the 200 Mammals Project, which will compare human DNA to that of 199 other mammals. It will help the researchers understand which core set of genes is so important that they’ve been preserved in us and other animals. And I’m involved with the Vertebrate Genomes Project to sequence all 66,000 living vertebrate species. These genomes reveal many interesting things about the history of an animal population, its patterns of migration and if it’s hybridized with other species. This kind of genetic analysis is going to open an astounding new window on how life works.

In your review, you discussed the creation of a global wildlife biobank. Why is that important?

Although the San Diego Zoo has its Frozen Zoo, with more than 1,000 species, that’s a small fraction of the world’s biodiversity. There are about 26,500 threatened species that have been named — and more that haven’t been put on the list.

Next year there will be less biodiversity than there is now. So we ought to be banking while we can. It needs to be done in different countries so they have their own genetic resources banked.

You said that when you arrived at the Frozen Zoo 44 years ago, you could not have imagined growing whole animals from cells. What project would you love to see the Frozen Zoo take on in the next 44 years?

As species’ populations shrink, they also lose valuable versions of genes that were only present in certain animals. The gene pool becomes a gene puddle. I imagine that in the future, using banked material, we could restore genetic variation.

How would you do that?

We’d have to turn cells into animals. For example, you might take a female animal, living in the wild, and transfer an embryo with some useful genes into her.

Some might say that bringing back lost genes, or near-extinct species, is playing God.

But humans have been doing that for a long time: We’ve caused species extinctions. We’ve irretrievably changed their habitats. We’ve altered their behavior. We’ve intermixed them. We’ve altered their environments, their predators, their diseases. All of these things we have already done.

Using banked samples to promote diversity would give us a chance to change the natural world in a positive way.

Knowable Magazine is an independent journalistic endeavor from Annual Reviews.

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