, in order to evaluate their performance and provide valuable insight for end-users. This introduces a need for testing newly developed FPs under various experimental conditions including expression vectors, cell types, model organisms etc. However, there is mounting evidence supported by numerous studies that high molecular brightness does not always correspond to high intracellular brightness in cultured cells and in vivo 13, 14, 15, 16. According to FPbase ( ) 12 there are multiple available FPs with high molecular brightness, mostly emitting cyan, green, yellow, and red fluorescence. One of the major criteria when selecting a genetically encoded FP for most in vivo applications is fluorescence brightness, among others 11. For example, investigation of clonal expansion and tissue plasticity as well as neuronal tracing are heavily reliant on spectral diversity of multicolor strategies 9, 10. Spectral diversity of fluorescent proteins (FPs) provides a straightforward approach for multiplex imaging of different subcellular and cellular structures in cultured cells 1, 2, 3, 4 and various model organisms 5, 6, 7, 8. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs.ĭuring the past few decades, there is an increasing need for simultaneous high-content imaging of multiple subcellular and cellular structures in intact biological systems. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.Ĭopyright (c) 2007 John Wiley & Sons, Ltd.Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors.
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