RESUMO
Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.
Assuntos
Proteínas Luminescentes/metabolismo , Imagem Corporal Total , Processamento Alternativo , Animais , Células HEK293 , Xenoenxertos , Humanos , Proteínas Luminescentes/genética , Camundongos , Modelos Animais , Imagem Molecular/métodos , Sítios de Splice de RNA , Razão Sinal-Ruído , Imagem Corporal Total/métodos , Proteína Vermelha FluorescenteRESUMO
Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.
Assuntos
Proteínas Luminescentes/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/toxicidade , Xenopus laevis , Proteína Vermelha FluorescenteRESUMO
The purpose of the study was to elucidate whether hepatitis C virus (HCV) RNA was present in the blood of patients from a Moscow therapeutic-and-prophylactic institution with uncertain results of a study of anti-HCV that were obtained using the algorithm developed for mass screening. The two-stage scheme of testing the serum for anti-HCV, which was obligatory in accordance with the Order of the Ministry of Health of the Russian Federation, was supplemented by a study the samples having a low optical density samples and those with controversial results in the test systems with the expanded spectrum of detectable anti-HCV, the instructions of which comprises criteria for an uncertain result. Sera with uncertain results of anti-HCV tests were assessed by two polymerase chain reaction (PCR) techniques for HCV RNA. The cDNA fragments complementary to HCV RNA were detected in the sera obtained from two elderly persons in PCR when a signal was recorded in agarose gel. The other real-time PCR failed to detect RNA in none sera with an uncertain result as to anti-HCV.
