Paradee and Genome Editing Team Make First CRISPR-engineered Mouse at Iowa

Date: Thursday, October 16, 2014

Dr. Bill Paradee is the new director of the Mouse Genome Editing Core Facility. Dr. Paradee joins Norma Sinclair, Patricia Yarolem, and JoAnn Schwarting in the generation of transgenic and knockout mouse production, and is committed to expansion of the mouse genetic manipulation services offered by the Core.

One of those services is bringing the relatively new CRISPR-based technologies to making mouse knockouts, as well as manipulations that rely on homologous-directed recombination. Instead of editing embryonic stems cells and grafting those into blastocysts, CRISPR technology can be leveraged by directly injecting fertilized eggs. Animals developed from these eggs have a high frequency of the desired genetic change.

As a proof of principle, the Genome Editing Core team targeted the mouse tryosinase gene (Figure 1). Spontaneous mutations exist in the tyrosinase gene that render the normally black C57 line of mice albino. By targeting tyrosinase, an observable phenotype appears even before the mice are old enough to be genotyped. The Core has already generated several albino and several chimeric mice from their first CRISPR injections.

_{Pronuclear injection of CRISPR reagents directed against
Tyrosinase yielded pups with coat-color phenotypes.}

“We were all really excited about producing these albino mice” says Bill Paradee. “It would have taken us several months to design/build a target vector to target this (or any) gene via homologous recombination in embryonic stem cells, as well as to screen for correctly targeted clones and then inject those clones into blastocysts, to get to the stage we are at now. Using the CRISPR technology, we were able to generate the reagents for these injections in less than two weeks.” The Core is working with several investigators to generate conditional knockouts and knock-ins of point mutations using the CRISPR technology. They anticipate being able to offer a targeted transgenic service using CRISPR by the end of the year.

The facility now has all the design tools, reagents, and protocols established for genome editing, and are constantly on the lookout for improvements as they arise in the field. Investigators with questions about making genetically altered mice in general, or how they can use CRISPR technology, can contact the Core to help navigate this quickly-developing technology.

These approaches are in addition to an expanding repertoire of services.

These include:

Sperm Cryopreservation Service

Made possible by a grant from the Carver Trust. Cryopreservation of mouse sperm is offered to "store" lines of animals not currently needed, to reduce cage costs and cage space, or to provide secure stock in case of contamination or catastrophic event.

Transgenic Mice via Pronuclear Injection

Transgenic animals are produced by injecting a DNA construct directly into the pronucleus of a newly fertilized mouse egg. The core acts to provide both state of the art equipment and expertise for the generation of genetically altered mouse models.

Genotyping

We perform project-specific genotyping using the Polymerase Chain Reaction Assay. The assay can be either designed by the investigator or by the Core.

Mouse embryo rederivation

Animals that come to the University of Iowa from other labs have unknown health risks and cannot be allowed into our “clean” facilities. We offer a rederivation service using embryos isolated from “dirty” mice which are then implanted in to “clean” mice in our facility.

Blastocyst Injection