Dataset Information

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

ABSTRACT: We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

SUBMITTER: Trinh le A

PROVIDER: S-EPMC3219234 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

Trinh Le A le A Hochgreb Tatiana T Graham Matthew M Wu David D Ruf-Zamojski Frederique F Jayasena Chathurani S CS Saxena Ankur A Hawk Rasheeda R Gonzalez-Serricchio Aidyl A Dixson Alana A Chow Elly E Gonzales Constanza C Leung Ho-Yin HY Solomon Ilana I Bronner-Fraser Marianne M Megason Sean G SG Fraser Scott E SE

Genes & development 20111101 21

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrin ...[more]

PMID: 22056673

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Project description:PurposeTo establish a workflow to visualize the surgical anatomy in 3D based on histological data of eyes of experimental animals for improving the planning of complex surgical procedures.MethodsFour C57BL/6J wild-type(wt) mouse eyes, three Brown Norway rat eyes and four Chinchilla Bastard rabbit eyes were enucleated and processed for standard histology with serial sections and hematoxylin and eosin staining. Image stacks were processed to obtain a representation of the eye anatomy in 3D. In addition, virtual image stacks and 3D point clouds were generated by processing sagittal sections of eyes with stepwise 180° rotation and projection around the eye axis to construct a rotationally symmetric 3D model from one single sagittal section.ResultsSerial sections of whole eyes of mice, rats and rabbits showed significant artifacts interfering with a practical image stack generation and straightforward 3D reconstruction despite the application of image registration techniques. A workflow was established to obtain a 3D image of the eye based on virtual image stacks and point cloud generation by rotation of a single sagittal section of the eye around the symmetry axis. By analyzing the tissue shrinkage during histological processing true biometric reconstructions of the eyes were feasible making the resulting model usable for 3D modeling and simulation, e.g. for planning of complex surgical procedures in different species.ConclusionBecause serial sections of the eye with standard histological protocols yielded too many artifacts for a straightforward 3D visualization we reconstructed a pseudorealistic 3D model based on virtual image stacks and point cloud generation calculated from a single sagittal section of the eye. Such a model detailing microscopic structures of the whole eye will allow for a specific planning of surgical procedures in small animal eyes in order to prevent surgical complications in a very early stage of an experiment and it will support the design and development of complex intraocular implants. It will therefore be helpful in surgical teaching and improve laboratory animal welfare by an expected reduction of experimental animal numbers. Further processing including integration of mechanical tissue properties is needed to convert these 3D models into a practical virtual reality teaching and simulation platform for eyes of several species.

Dataset Information

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

Publications

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

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