Generation of Transgenic Medaka Oryzias curvinotus (Nichols & Pope, 1927) Carrying a Cyan Fluorescent Protein Gene Driven by Alpha Actin Promoter

The study aimed to produce fluorescent protein transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) as a novel strain of ornamental fish. These fish were produced by transferring a plasmid consisting of a fluorescent reporter gene and a strong promoter into one-cell stage embryos. For this purpose, myosin light chain 2, but not other promoters, was mainly used. The study also evaluated the stability of the transgenic medaka germline acquiring vivid fluorescent phenotypes via the transgenesis of the cyan fluorescent protein (CFP) gene under the control of O. curvinotus skeletal alpha-actin (OCacta) promoter. The pOCacta-CFP plasmid, containing a OCacta promoter and CFP reporter gene, was transferred into the one-cell stage of O. curvinotus embryos by a microinjection technique. As a result, 36 of 1386 microinjected O. curvinotus embryos exhibited CFP signals in their trunks. The expressed CFP signals in O. curvinotus embryos and adults were detected under a microscope using a green fluorescent protein (GFP) filter (450–490 nm wavelength), and blue LED light (400–450 nm wavelength). Five O. curvinotus founders showing clear CFP signals were selected and crossed with non-transgenic counterparts to produce subsequent generations. Among strains, the frequency of germline transmission from founder to F1 was highly variable. Only two of the five founders successfully pass the transgene to the F1 generation. At present, the progeny of subsequent generations is being produced and tested for the expression of CFP signals, and therefore, stable lines are ongoing.

Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering

Cyan fluorescent proteins (CFPs) are variants of green fluorescent proteins in which the central tyrosine of the chromophore has been replaced by a tryptophan. The increased bulk of the chromophore within a compact protein and the change in the positioning of atoms capable of hydrogen bonding have made it difficult to optimize their fluorescence properties, which took approximately 15 years between the availability of the first useable CFP, enhanced cyan fluorescent protein (ECFP), and that of a variant with almost perfect fluorescence efficiency, mTurquoise2. To understand the molecular bases of the progressive improvement in between these two CFPs, we have studied by incoherent neutron scattering the dynamics of five different variants exhibiting progressively increased fluorescence efficiency along the evolution pathway. Our results correlate well with the analysis of the previously determined X-ray crystallographic structures, which show an increase in flexibility between ECFP and the second variant, Cerulean, which is then hindered in the three later variants, SCFP3A (Super Cyan Fluorescent Protein 3A), mTurquoise and mTurquoise2. This confirms that increasing the rigidity of the direct environment of the fluorescent chromophore is not the sole parameter leading to brighter fluorescent proteins and that increased flexibility in some cases may be helpful.

Crystal structure of the cyan fluorescent protein Cerulean-S175G

Enhanced cyan fluorescent protein (ECFP) was derived fromAequorea victoriagreen fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that ofavGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations calledECFPandCerulean. In this study, Cerulean-S175G was revealed to adopt only theCeruleanconformation, while Cerulean has been reported to adopt both theECFPand theCeruleanconformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt theECFPconformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145–148 of β-strand 7.

PCl3-mediated synthesis of green/cyan fluorescent protein chromophores using amino acids

An efficient synthesis of green and cyan fluorescent protein chromophores from l-tyrosine and l-tryptophan using PCl3 has been successfully developed.