In Vitro Screen to Obtain Widespread, Transgenic Expression in the Mouse
Adapted from Manipulating the Mouse Embryo, 3rd edition, by Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. CSHL Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
This protocol describes the steps for creating widespread (ubiquitous-like) transgenic expression using embryonic stem (ES) cell-mediated transgenesis. The use of ES cells results in mostly a single-copy or low-copy-number integration. Several hundred different integration sites can occur in a single experiment. The in vitro expression of these clones can be assayed before a decision is made about introducing the candidate cell line(s) into the mouse. The Cre excision-conditional transgene vector has an easy readout for the nature of transgene expression. Upon introduction into ES cells, such transgenes will express βgeo only, which confers G418 resistance on the cells. Staining the cells for lacZ is an easy way to characterize expression in undifferentiated and in vitro-differentiated ES cells.
