Dissecting the Role of the Ccr4–Not Deadenylase Complex in Pluripotency and Differentiation

In this chapter, we describe methods and assays to examine the role of the Ccr4–Not mRNA deadenylase complex in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. We present the following procedures: sgRNA design and cloning, the culture of mouse ESCs, transfection and colony picking, genotyping, generation of Cnot3 conditional deletion ESCs, assessment of Cnot3 deletion efficiency, and examination of the impact on ESC maintenance and differentiation. The above approach and protocols can also be readily applied to study other Ccr4–Not subunits in ESC fate regulation, thereby facilitating the systematic dissection of Ccr4–Not function in stem cells and development.