Efficient generation of endogenous protein reporters for mouse preimplantation embryos

Abstract

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals. However, 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. We present two approaches for higher throughput production of endogenous protein reporters. Our first approach 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, eliminating 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. However, we report a threshold of detection using this approach. By contrast, the V5 epitope enabled high efficiency and higher sensitivity protein reporting. We describe complementary advantages and prospective applications of these two approaches. HighlightsO_LISplit fluorescent protein for in vivo protein localization in living embryos C_LIO_LIV5 tagging for in vivo localization of low abundance proteins C_LIO_LIBypassing the need for founder mouse lines for preimplantation studies C_LIO_LIGuidelines and strategies for implementation and prospective applications C_LI