Efficient generation of endogenous protein reporters for mouse development.

Abstract

ABSTRACTFluorescent proteins and epitope tags can reveal protein localization in cells and animals, yet the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. Here, we present two strategies for higher throughput production of endogenous protein reporters in mice, focusing on the blastocyst model of development. Our first strategy makes use of a split fluorescent protein, mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest, was highly efficient in embryos, potentially obviating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. Our second strategy achieves in-frame knock-in of a relatively small protein tag, which provides high efficiency and higher sensitivity protein reporting. Together, these two approaches provide complementary advantages and enable broad downstream applications.