Multicolor fluorescent protein

A NOVEL MULTICOLOR FLUORESCENT PROTEIN FROM THE SOFT CORAL SCLERONEPHTHYA GRACILLIMA KUEKENTHAL... 

A Novel Multicolor Fluorescent Protein from the Soft Coral S. gracillima Kuekenthal 313... 

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

In the coming years we can look forward to widespread use of confocal FRAP as well as the implementation of new techniques which enhance the imaging and/or data acquisition of these experiments. Already gaining momentum is the use of multicolor FRAP which allows for the simultaneous analysis of two or more fluorescent molecules that have minimal to no spectral overlap (such as eGFP and mCherry). The use of two-colored FRAP could be used to study the dynamics of two proteins in different but overlapping cellular compartments and of two proteins of the same complex. Moreover, mutant proteins could be directly compared with cvild-type versions within the same cell (20). 

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