IOS EDGE: Rapid and Efficient Gene Editing of Amphibians Through Nuclear Transfer from Engineered Cell Lines

Oklahoma Medical Research Foundation, Oklahoma City OK

Investigators

Abstract

Amphibians are outstanding models for vertebrate physiology and development. Among their many advantages are inexpensive housing, production of large clutches of eggs and embryos, embryonic development outside the mother, ease of experimental manipulation, and an extensive existing literature characterizing many aspects of their biology. However, modern genetic approaches in amphibians are extremely limited by their long breeding cycles and the difficulty of rearing and maintaining lines of mutant animals. The approach detailed in this project overcomes the limitations by proposing gene editing in amphibian cultured cell lines where genetic manipulations are much simpler. Cells with the desired genetic modification are then used to generate mutant animals. Thus, with no breeding, the researcher obtains specific desired mutant animals in a very short time. Unlike animals, cell lines with desired mutations can be frozen and stored indefinitely. This approach has the potential to revolutionize genetic manipulation in amphibians, making it fast, efficient, and powerful. The project is intimately tied to the mission of the National Xenopus Resource (NXR) located at the Marine Biological Laboratory (MBL) in Woods Hole, MA in assisting scientists to make use of amphibian models, to develop new technologies, and to disseminate these tools to the scientific community. The NXR also has an important function in educating students and teachers at all levels. The NXR carries out its mandates through participation in the many courses held at the MBL, by hosting hands-on workshops in genetic manipulation of amphibians, and through visits by scientists, students and educators. Despite their many advantages for studies in fields such as development, regeneration and vertebrate physiology, amphibians lack practical methods for generating F0 (first generation) embryos with precisely defined, biallelic mutations. The long-term goal of the project is to make Xenopus and other amphibians vastly more powerful systems by simplifying the generation of precisely defined mutant animals. Another, overarching goal is to generalize this approach to enable the production of F0 mutants in a wide variety of vertebrate and invertebrate species, particularly in emerging models with little or no conventional genetics. The specific objectives of this project are to develop reliable methods for generating X. laevis, X. tropicalis, and axolotl cell lines with specific mutations, primarily using CRISPR-Cas9 technology. These cell lines can be cloned, fully characterized to define the specific genetic change, then stored frozen indefinitely. Nuclei from these cells will then be transferred to enucleated eggs and used to generate F0 mutant amphibian embryos. The assembled team combines expertise in amphibian cell culture and embryology and is particularly well-suited for carrying out this project and for rapid and efficient transmission of the technology to the scientific community.