Conservation cloning is a tool of last resort. Its most promising uses are either for species that are descended from just a handful of individuals, known as a genetic bottleneck, or species that are in danger of going extinct now. Even though Elizabeth Ann's birth demonstrates cloning's potential hope for at-risk mammals, it’s a different story when it comes to avian conservation.
“We’ve been asked a few times now, ‘when is this going to happen for birds?’,” says Ben Novak, a lead scientist with Revive & Restore, the organization that spearheaded the black-footed ferret cloning efforts. The answer, Novak says, is probably never—cloning likely isn’t possible in birds.
To understand why, it’s important to know how cloning works. There’s more than one way to get a clone (including, for instance, identical twins), but the technique made famous by Dolly the sheep and used to create Elizabeth Ann is called “somatic cell nuclear transfer.” This process involves swapping out the genetic information of an egg cell and replacing it with the DNA of a donor individual. First, the researchers take the egg cell and place it under a microscope. Then, they zero in on its nucleus, which is the part of the cell that contains the vast majority of its genetic information. They carefully suck that nucleus out, leaving the egg cell empty of its genetic code. This means that the egg is now a blank slate ready to be filled with a new set of instructions.
The next step is to insert either a body cell (also known as a somatic cell) or its nucleus from the individual they want to clone, which contains the donor’s genetic material. Once the new cell and its instructions are inside the egg, researchers encourage the two to fuse with a jolt of electricity. If everything has gone right, this tricks the egg cell into thinking it has been fertilized, and it will start to divide and multiply, forming an embryo. After growing for a while in a petri dish, the resulting embryo can be implanted in the uterus of a surrogate mother, who will give birth to a clone.
In practice, only a fraction of these nuclear transfers result in viable embryos that go on to become adult animals. Even so, a lot of mammals—even wild species like deer, gray wolves, and macaques—have been cloned in this way since Dolly the sheep proved it was possible back in 1996. But there has never been a single cloned bird.
There are a few reasons why birds have been impossible to clone so far. The biggest hurdle? “It’s what you had for breakfast,” says Tom Jensen, a reproductive scientist with the San Diego Zoo Wildlife Alliance. “The egg yolk.”
That nucleus-swapping trick is easy enough to do with mammal egg cells, which range from about one to three human hairs’ width in diameter, Jensen explains. These little eggs are the perfect size to slide under a microscope, where scientists can get down to a microscopic level and locate the tiny DNA-containing nucleus. But in birds, the egg cell—what we call the yolk—is a lot bigger. And the nucleus holding all of the bird's DNA is a miniscule white dot in the middle of that yolk, which the embryo will eventually grow out of.
The yolk is basically a bird embryo's packed lunch from mom, providing it with all the nutrients it needs to eventually grow into a baby bird ready to hatch. The arrangement is an efficient one, but it also presents two major issues for scientists trying to clone a bird. First, the yolk's size prevents it from fitting under a microscope to conduct the necessary work. And second, even if scientists could inspect the yolk on a microscopic level, finding that tiny nucleus floating somewhere in the yolk is extremely challenging. Novak has described the process as akin to "looking for a white marble in a pool of milk."
Another issue with eggs is that once the yolk leaves the ovary, it is always on the move. During the typical cloning process with a mammal, researchers can just stick the embryo in a surrogate mother’s uterus and let it grow. Birds have no such incubation chamber. After the yolk forms, it’s dropped into something called the oviduct, where it tumbles down an assembly line that coats it with first the egg white, and then the shell membrane. There is no uterus equivalent in which to stick a bird clone embryo.
“It’s technically much easier in a mammal than it would be in a bird,” Jensen says. “So, I don’t think it’ll ever be anything we can use for bird conservation.”
Currently, without the ability to cryopreserve the cells of bird species and clone them later, there is no scientific failsafe for birds like there is for mammals in case of genetic bottlenecks or critical endangerment. However, Jensen is hoping a different emerging genetic technology could perform a similar role for endangered bird species. This one focuses not on creating an entire clone, but on altering what kinds of chicks an individual is producing. That requires researchers to focus on birds' testes and ovaries, also called the gonads. “We don’t really care that [the host species] looks like the animal, what we care about is that its gonads are identical to the animal that we’re cloning, right?” Jensen says. “In birds, we can kind of clone the gonad.”
The bird answer to cloning hinges on something called “primordial germ cells,” or PGCs. That’s basically a fancy term for the cells that are destined to become sperm and egg cells. Cloning a mammal involves inserting the DNA of one individual into one egg, and producing one offspring that is the genetic replica of the donor. With PGC technology, the goal is to create a surrogate with gonads that contain the DNA of another species. Reseearchers refer to these hybrid animals as chimeras.
To make a chimera, researchers carefully insert PGCs from a donor into a host embryo as it is developing. The PGCs then migrate down to the host embryo’s gonads. If everything goes to plan, the host will grow up to become an adult that produces sperm or eggs containing the DNA of the donor.
So, for instance, you might have a domestic rooster host who produces sperm with the DNA of a Greater Prairie-Chicken. If the rooster were mated with a female Greater Prairie-Chicken, the pair could then produce Greater Prairie-Chicken chicks. This approach has the benefit that one host parent could theoretically produce many offspring with the DNA of a donor over the course of its lifetime, rather than the single individual that would be produced via cloning. And, as long as researchers can cryopreserve these PGCs, this can still offer the benefit of bringing back DNA from a donor that died long ago.
The catch is that PGC technology is much trickier than cloning, with more steps that could potentially go wrong, so it’s still in development. Researchers have made some progress, however, that makes Jensen and Novak hopeful that the technology could one day be used for conservation.
Most of the research thus far has involved domestic chickens. Several groups of researchers have successfully transferred PGCs from one breed of chicken into another, producing offspring with donor DNA. Researchers have also used the technique to successfully produce Maya Ducks, Korean Pheasants, and even Houbara Bustards by mating chimera domestic chicken males to the females of those species.
Jensen and his team, for their part, are looking into whether stem cells from dead individuals can be used for this technique, and trying to figure out how far apart the donor and the host species can be genetically. So far, any cells his lab has experimentally introduced to chicken embryos have migrated to the gonads like they’re supposed to. “Which is a really, really good sign for working across multiple species—even distantly related species,” Jensen says. “Although we have not yet hatched any chickens to verify functional sperm production.”
Revive & Restore, meanwhile, is in the process of producing interspecies chimeras with conservation in mind. More specifically, they’re in the preliminary stages of getting domestic chickens to produce Greater Prairie-Chicken sperm. Rosemary Walzem, a researcher with Texas A&M University, is currently leading these efforts. Her lab hosts a flock of Greater Prairie-Chickens, and this spring her team is hoping to carefully extract some primordial germ cells (a tricky task in its own right) to eventually try to introduce into domestic chickens. The idea is to perfect the process with a species that is closely related to domestic chickens, then apply what they learn to related species that are endangered—or even extinct.
Eventually, this technology might help to improve the genetic diversity of species like California Condors or Whooping Cranes that are descended from just a few individuals. “Pretty much any breeding program where you could go in and bring an individual back from decades later would really benefit from that diversity injection,” Jensen says.
Ideally, this technology would never be necessary. The best approach to conservation is to keep species plentiful and their habitats and ecosystems intact. But it could be a valuable tool in worst-case scenarios. Jensen and Novak say that they ultimately hope the technology will be developed to the point that researchers can capture wild birds with mist nets, extract their primordial germ cells, and then release them. The cells could then be stored away, or they could immediately be placed in a host to reproduce more of the species without having to take a wild population into captive breeding. But that vision is still a long way off, both admit. “Our goals are very ambitious,” Novak says. “But we think they’re doable.”