Dual transgene strategy for live visualization of chromatin and plasma membrane dynamics in murine embryonic stem cells and embryonic tissues.
To simultaneously follow multiple subcellular characteristics, for example, cell position and cell morphology, in living specimens requires multiple subcellular labels. Toward this goal, we generated dual-tagged mouse embryonic stem (ES) cells constitutively expressing differentially localized, spectrally distinct, genetically encoded fluorescent protein fusions. We have used human histone H2B fusions to fluorescent proteins to mark chromatin. This provides a descriptor of cell position, division, and death. An additional descriptor of cell morphology is achieved by combining this transgene with select lipid-modified fluorescent protein fusions that mark the plasma membrane. Using this strategy, wewere able to live image cellular dynamics in three dimensions over time both in cultured ES cells and in mouse embryos generated using dual-tagged ES cells. This study, therefore, presents the feasibility of applying multiple spectrally and subcellularly distinct fluorescent protein reporters for live imaging studies in ES cells and mouse embryos. Furthermore, the increasing availability of spectral variant fluorescent proteins along with the development of methods that permit improved spectral separation now facilitate multiplexing of fluorescent reporters to provide readouts of a variety of anatomical and physiological behaviors simultaneously in living specimens.
Sloan-Kettering Institute, New York, New York, USA.
Sonja
S
Nowotschin
Guy S
GS
Eakin
Anna-Katerina
AK
Hadjantonakis
D000818:Animals; D002449:Cell Aggregation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002678:Chimera; D002843:Chromatin / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005240:Feasibility Studies; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics*; D000097573:Red Fluorescent Protein
D002843:Chromatin; D049452:Green Fluorescent Proteins; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein