Dataset Information

Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.

ABSTRACT: Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-green fluorescent protein mouse was found to faithfully recapitulate the expression pattern of the Prox1 gene in lymphatic endothelial cells and other Prox1-expressing organs, and enabled us to conveniently visualize detailed structure and morphology of lymphatic vessels and networks throughout development. Our data demonstrate that this novel transgenic mouse can be extremely useful for detection, imaging, and isolation of lymphatic vessels and monitoring wound-associated lymphangiogenesis. Together, this Prox1-green fluorescent protein transgenic mouse will be a great tool for the lymphatic research.

SUBMITTER: Choi I

PROVIDER: S-EPMC3037757 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.

Choi Inho I Chung Hee Kyoung HK Ramu Swapnika S Lee Ha Neul HN Kim Kyu Eui KE Lee Sunju S Yoo Jaehyuk J Choi Dongwon D Lee Yong Suk YS Aguilar Berenice B Hong Young-Kwon YK

Blood 20101020 1

Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-gree ...[more]

PMID: 20962325

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Project description:Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based on Escherichia coli surface-displayed streptavidin (SavSD hereafter). Through the binding of a ruthenium-pianostool cofactor to SavSD, an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase of in vivo activity was observed for SavSD S112T-K121G compared to the wild-type SavSD (wt-SavSD). Finally, the best performing Sav isoforms were purified and tested in vitro (SavPP hereafter). For SavPP S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-SavPP. To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of SavSD was determined. For this purpose, crosslinking experiments of E. coli cells overexpressing SavSD were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that SavSD is most likely displayed as a monomer on the surface of E. coli. We hypothesize that the difference between the in vivo and in vitro screening results may reflect the difference in the oligomeric state of SavSD vs soluble SavPP (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using E. coli surface display.

Project description:BackgroundDespite greatly renewed interest concerning meningeal lymphatic function over recent years, the lymphatic structures of human dura mater have been less characterized. The available information derives exclusively from autopsy specimens. This study addressed methodological aspects of immunohistochemistry for visualization and characterization of lymphatic vessels in the dura of patients.MethodsDura biopsies were obtained from the right frontal region of the patients with idiopathic normal pressure hydrocephalus (iNPH) who underwent shunt surgery as part of treatment. The dura specimens were prepared using three different methods: Paraformaldehyde (PFA) 4% (Method #1), paraformaldehyde (PFA) 0.5% (Method #2), and freeze-fixation (Method #3). They were further examined with immunohistochemistry using the lymphatic cell marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and as validation marker we used podoplanin (PDPN).ResultsThe study included 30 iNPH patients who underwent shunt surgery. The dura specimens were obtained average 16.1 ± 4.5 mm lateral to the superior sagittal sinus in the right frontal region (about 12 cm posterior to glabella). While lymphatic structures were seen in 0/7 patients using Method #1, it was found in 4/6 subjects (67%) with Method #2, while in 16/17 subjects (94%) using Method #3. To this end, we characterized three types of meningeal lymphatic vessels: (1) Lymphatic vessels in intimate contact with blood vessels. (2) Lymphatic vessels without nearby blood vessels. (3) Clusters of LYVE-1-expressing cells interspersed with blood vessels. In general, highest density of lymphatic vessels were observed towards the arachnoid membrane rather than towards the skull.ConclusionsThe visualization of meningeal lymphatic vessels in humans seems to be highly sensitive to the tissue processing method. Our observations disclosed most abundant lymphatic vessels towards the arachnoid membrane, and were seen either in close association with blood vessels or remote from blood vessels.

Project description:BackgroundAlthough the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.ResultsA high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.ConclusionsMany of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

Dataset Information

Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.

Publications

Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.

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