Production of large, defined genome modifications in rats by targeting rat embryonic stem cells.

Production of large, defined genome modifications in rats by targeting rat embryonic stem cells.

Stem cell reports (2022-12-17)

Jeffrey Lee, Jingjing Wang, Roxanne Ally, Sean Trzaska, Joseph Hickey, Alejo Mujica, Lawrence Miloscio, Jason Mastaitis, Brian Morse, Janell Smith, Amanda Atanasio, Eric Chiao, Henry Chen, Adrianna Latuszek, Ying Hu, David Valenzuela, Carmelo Romano, Brian Zambrowicz, Wojtek Auerbach

PMID36525967

ABSTRACT

Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb. We report here the generation of germline-competent rESC lines from several rat strains. These rESC lines maintain their potential for germline transmission after serial targeting with bacterial artificial chromosome (BAC)-based targeting vectors, and CRISPR-Cas9 cutting can increase targeting efficiency. Using these methods, we have successfully replaced entire rat genes spanning up to 101 kb with the human ortholog.

MATERIALS

Product Number

Brand

Product Description

P8340

Sigma-Aldrich

Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution