The laboratory opossum is the only marsupial that is available in large numbers for biomedical research. It is a
unique or specialized model for research on many human diseases and developmental processes, as well as
for comparative biology and comparative genomics purposes. The PI maintains by far the largest breeding and
research colony of this species in the world. The most critical barrier to fulfilling the research potential of the
laboratory opossum is the lack of success of anyone in the US in establishing gene-editing procedures for this
species. A Japanese group recently overcame the technological impediments to gene editing of this species,
and succeeded in knocking out the tyrosinase (Tyr) gene in random-bred opossums. The implementation and
optimization of their methods at the PI’s laboratory will pave the way for achieving the long-term objective,
which is to establish a national research resource that will efficiently and economically create gene-
edited opossums required by US investigators to address important biomedical questions that this
laboratory animal is uniquely suited to address. While the volume of research with this species at any single
institution cannot justify the cost of establishing a large colony and gene editing technologies, a centralized
resource will be capable of serving all of the national needs at minimal cost. The specific aims are 1) to
establish expertise and proof-of-principle in our laboratory by targeted disruption of Tyr in a fully inbred
strain of laboratory opossums, and 2) to conduct targeted disruption of the phosphatase and tensin
homolog gene (Pten) in opossums of the same inbred strain. Knockout opossums will be created by applying
CRISPR-Cas9 technology to opossum embryos collected 30 - 34 hours after copulation, after which the egg
shell becomes too hard to penetrate. Penetration will be enhanced via the use of a piezoelectric actuator.
Confirmation of knockout genotype will be established by genomic DNA analysis of progeny weaned from the
treated embryos, and from subsequent generations of animals produced from those progeny. Pten -/-
homozygotes are expected to embryonic lethal, but heterozygotes are expected to serve as new model
in comparative medicine research on Autism Spectrum Disorder. We plan to establish a research project
on that topic beyond the 2-year project period, after a sufficient number of +/- animals have been produced.
Some examples of other research programs that will be made possible or highly enhanced by the creation of
specific knockouts are provided in the letters of support from investigators who are eager to collaborate.
Examples include the use of Pten +/- opossums in conjunction with Pten knockout opossum iPSCs (+/- and -/-)
from the same inbred strain for comparative research on in vivo and ex vivo neurodevelopmental processes
(Whitworth), the use of Pten +/- opossums to investigate mechanisms involved in auditory function and
regeneration (Walters), identification of genes that confer remarkable resistance of opossums to snake venom
(Sanchez), and function of T cell subsets, of which marsupials have one that is not found in eutherians (Miller).