Establishment And Application Of Polycistronic Expression System In C. Elegans And Super-Resolution Imaging Of DCV Related Protein In INS-1Cell

This thesis includes two parts:The first part mainly focuses on the establishment and application of polycistronicexpression system in C. elegans.(chapter1,2,3)Heterologous gene(s) can be expressed in organisms or cells by transgenic technique.“Multi-gene expression under the control of a single promoter” can also be called as“polycistronic expression strategy”. It is broadly used in biological research and genetherapy. Construction of multi-gene expression vector is the basis of its application.Conventional approaches for multicistronic expression are derived from internalribosome entry sites (IRES) or2A self-cleaving peptide. Unfortunately, multicistronicexpression vectors using IRES cannot be applicable for expression in an important modelsystem: Caenorhabditis elegans. While the disadvantage of multicistronic expressionstrategy using2A self-cleaving peptide is that there will be an additional peptide adding tothe C terminus of the upstream protein. It may disturb the structure and function of thisprotein.Here we describe a multi-gene expression strategy based on trans-splicingmechanism of C. elegans. Two fluorescent proteins linked by a trans-splicing sequencecan be expressed under a single promoter in body wall muscle cells or nervous system ofC. elegans. We also studied the properties of this polycistronic expression system. Weshowed that at least five proteins could be generated from a single transcription unit. Theexpression efficiency of each protein is independent of the distance between the gene andpromoter.We displayed some applications of this polycistronic expression system in C. elegans:1, The expression output of protein can be greatly improved by expression of its cistronictandem repeat.2, This strategy ensures the gene of interest and reporter gene to betranslated from a single bicistronic mRNA, which facilitates the selection of transformantsand confirms the cell-specific gene expression.3, Particularly, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins withdifferent maturation times to monitor the activity of targeted promoters in C. elegans.4, Invivo ratiometric calcium imaging was achieved by bicistronic expression of G-CaMP2.0and mKate2.5, We also constructed a series of cloning vector, to facilitate the study in ourlab.The second part is about the application of new developed fluorescent proteins influorescent imaging microscopy.(chapter4,5)In chapter4, we performed dual-color PALM(photoactivated localization microscopy)using photoswitchable fluorescent protein mGeosF and photoactiviated fluorescent proteinPATagRFP to study the constitution and exocytosis mechanism of dense core vesicle inINS-1cells.The localization or distribution information of proteins is important for understandingits function. The spatial resolution of traditional optical microscopy is about200nmaccording to the Abbe wave theory. In recent years, to overcome the diffraction barrier, anumber of “super-resolution” fluorescence microscopy techniques have been invented,such as stimulated emission depletion microscopy (STED), saturatedstructured-illumination microscopy (SSIM), photoactivated localization microscopy(PALM), and stochastic optical reconstruction microscopy (STORM). Spatial resolution inall three dimensions has been largely improved using these super-resolution microscopytechniques. The observation of previously unresolved details of cellular structures hasdemonstrated the great promise of super-resolution fluorescence microscopy in elucidatingbiological processes at the cellular and molecular scale.The most popular strategies used to study the constitution and composition of DCVare mass spectrometry(MS) and electron microscopy(EM). Comprehensive proteomiccharacterization of the categories of proteins in DCV can be provided by nano-HPLC ChipMS/MS tandem mass spectrum etry, however, the localization information of theseproteins in DCV remains unknown. Electron microscopy technique can be used toevaluate the size distribution and structure of DCVs in cells, however, we still cannotobtain the localization information of related proteins.Here, the DCV related proteins were labeled with photo-switchable orphoto-activated fluorescent proteins such as mGeosF and PATagRFP to perform PALM super resolution imaging in INS-1cells.We can get the2D(two dimension) localization information of these proteins atspatial resolution of~20nm. Size distribution and structure of DCVs can also be obtainedfrom the PALM images.It establishes a solid foundation for further studying the constitution and exocytosismechanism of DCV using3D PALM or new developed imaging technique: correlativemicroscopy of PALM and EM.In chapter5, we presented a novel fluorescent protein pair of mKate2and mAmetrinefor dual-color two-photon imaging.Dual-color two-photon laser scanning microscopy is a useful method forsimultaneously studying the expression, localization and trafficking of two differentproteins in tissues. Because most two-photon microscopes only use a single wavelengthexcitation laser, simultaneously exciting multiple fluorescent proteins remains a challenge.Here, we present mAmetrine and mKate2, which can be used as a novel fluorescentprotein pair in dual-color two-photon imaging by taking advantage of the large Stokesshift of mAmetrine and high brightness of mKate2. Both proteins have high two-photonabsorption efficiencies and can be simultaneously excited at an optimal wavelength of765nm. Dual-color two-photon imaging using this protein pair is highly effective in livingcells.