Although kangaroos and koalas are better known, researchers who study marsupials often use opossums in laboratory experiments because they are smaller and easier to care for. Gray short-tailed opossums, the species used in the study, are related to North American white-eyed opossums, but are smaller and do not have a sac.
Researchers from Riken used CRISPR to remove or knock out a gene that encodes pigment production. Targeting this gene meant that the experiments, if successful, would be obvious at first glance: opossums would be albino if both copies of the gene were ejected, and mottled or mosaic if one copy were deleted.
The resulting litter included one albus opossum and one mosaic opossum (picture above). The researchers also bred the two of them, resulting in a litter of a completely albino possum, showing that the color is an inherited genetic trait.
The researchers had to cross several obstacles to edit the possum genome. They first had to determine the timing of hormone injections to prepare the animals for pregnancy. Another challenge was that the egg sacs develop a thick layer around themselves, called the mucoid shell, soon after fertilization. This makes it difficult to inject CRISPR treatment into cells. In the first attempts, the needles would either not penetrate the cells or damage them so that the embryos could not survive, Kiyonari says.
The researchers realized that it would be much easier to inject at an earlier stage, before the coating around the egg becomes too tight. By changing when the lights were turned off in the labs, the researchers mating the opsums later in the evening to get the eggs ready to work in the morning, about a day and a half later.
The researchers then used a tool called a piezoelectric drill, which uses an electric charge to make it easier to penetrate the membrane. This helped them inject the cells without damage.
“I think it’s an amazing result,” he says Richard Behringer, a geneticist at the University of Texas. “It simply came to our notice then. Now is the time to do biology, ”he added.
Oposes have been used as laboratory animals since the 1970s, and researchers have been trying to edit their genes for at least 25 years, says VandeBerg, who began trying to create the first laboratory colony of possums in 1978. They were also the first marsupial to get their genome completely sequenced, 2007
Comparative biologists hope that the ability of genetic modification of the opossum will help them learn more about some unique aspects of marsupial biology that have yet to be decoded. “We’ve found genes and baggy genomes that we don’t have, so that creates a bit of a mystery about what they do,” he says. Rob Miller, an immunologist from the University of New Mexico, who uses possums in his research.