While kangaroos and koalas are better known, researchers who study marsupials often use possums in lab experiments because they are smaller and easier to maintain. Gray short-tailed possums, the species used in the study, are related to North American white-faced opossums, but they are smaller and have no pouch.
Riken researchers used CRISPR to suppress or eliminate a gene that codes for pigment production. Targeting this gene meant that if the experiments worked, the results would be obvious at a glance: possums would be albinos if both copies of the gene were removed, and mottled, or mosaic, if only one copy was removed.
The resulting litter included an albino opossum and a mosaic opossum (pictured above). The researchers also bred the two, resulting in a litter of fully albino possums, showing that the coloring was an inherited genetic trait.
Researchers had to overcome a few hurdles to modify the opossum genome. First, they had to determine the timing of the hormone injections to prepare the animals for gestation. The other challenge was for the marsupial eggs to develop a thick layer around them, called a mucoid shell, soon after fertilization. This makes it more difficult to inject CRISPR processing into cells. In their first attempts, the needles did not penetrate or damage cells, so the embryos could not survive, says Kiyonari.
The researchers realized that it would be much easier to give the injection at an earlier stage, before the coating around the egg got too hard. By changing when the lights went out in the labs, the researchers got the opossums to mate later in the evening so that the eggs were 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 electrical charge to more easily penetrate the membrane. This helped them inject the cells without damaging them.
“I think this is an incredible result,” said Richard behringer, geneticist at the University of Texas. “They have shown that it can be done. Now is the time to do biology, ”he adds.
Opossums have been used as laboratory animals since the 1970s, and researchers have attempted to alter their genes for at least 25 years, says VandeBerg, who began trying to create the first lab opossum colony in 1978. They were also the first marsupials to have their fully sequenced genome, in 2007.
Comparative biologists hope the ability to genetically modify opossums will help them learn more about some of the unique aspects of marsupial biology that have yet to be decoded. “We find genes and marsupial genomes that we don’t have, which creates a bit of a mystery as to what they are doing,” explains Rob miller, an immunologist at the University of New Mexico, who uses opossums in his research.