Organ generation from knockedout rat blastocysts complemented with pluripotent stem cells

Abstract

Regeneration of human organs in domestic animal model would provide enough number of functional donor organs in transplantation therapy. Recent progresses in pluripotent stem cells and nuclease-based genome editing tools have set the stage for investigating the chimeric complementation approach to generate functional organs from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In this chapter, protocol for allogeneic or xenogeneic organ generation using knocked-out (KO) rat blastocysts and the rat or mouse ES/iPS cells is described. The protocol includes (1) the preparation of KO rat colony, (2) the preparation of rat or mouse ES/iPS cells, (3) the recovery of rat blastocysts, (4) the stem cell injection into blastocysts, (5) the embryo transfer into pseudopregnant recipient uteri, and (6) the genotyping and organogenetic analysis of chimeric offspring. The accumulation of basic and practical knowledge in the rodent model would be useful in improving therapeutic performance to regenerate 3D organs available for transplantation.