Microinjection and Oviduct Transfer Procedures for Rat Model Generation with CRISPR-Cas9 Technology.

ABSTRACT

Since the first knockout rat model was generated with zinc-finger nucleases (ZFNs) by Geurt's group in 2009, the demand for making targeted rat models has increased tremendously. The advent of the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system provides researchers with a more efficient method for producing modified animals, which has since then been developed and applied in rat. Since we established a rat model production system at our facility in 2014, we have consistently generated rat models. Due to differences in physiology and embryology between mouse and rat, species-specific protocols for superovulation conditions, microinjection, and embryo transfer (among others) are required. There are over 100 rat strains, and Sprague Dawley is one of the commonly used outbred strains in biomedical research. In this chapter, we describe in detail a range of topics including donor and recipient preparation, microinjection setup, CRISPR reagent preparation, and oviduct transfer procedures for making rat models in the Sprague Dawley background.