Creation of Knockout Laboratory Opossums

University Of Texas Rio Grande Valley, Edinburg TX

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT 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 the largest breeding and research colony of this species in the world. The most critical barrier to fulfilling the full 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 a random-bred stock. 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 is capable of efficiently and economically creating gene-edited opossums required by US investigators to address important biomedical questions that this laboratory animal is uniquely suited to address. The specific aims of the parent project 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 prior to 34 hours after copulation, when 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. The specific aim of the supplement is to conduct targeted disruption of the µ gene that encodes the µ polypeptide of the γµ T cell receptor dimer. All mammals have the αβ and γδ T cell receptors, but only marsupials have the µ gene and the γµ T cell receptor. Its function is not known. Creating a knockout of the µ gene will provide new insights into the mechanisms by which T cell receptors contribute to cellular immunity. This aim will be accomplished using the techniques that are being developed in the parent project. After the conclusion of the parent grant and the supplement, the animals with the disrupted µ gene will be bred to produce a stock that will be used for research on function of the γµ T cell receptor and its functional relationships with the αβ and γδ T cell receptors. Since T cell receptors are critical to the immune response to pathogens and to immunosurveillance for cancers, opossums lacking the γµ T cell receptor may play an important role in improving our understanding of mechanisms by which T cell receptors function, and ultimately in developing new strategies for preventing and treating infectious diseases and cancer.