20 research outputs found
Enhancer trapping in zebrafish using the <it>Sleeping Beauty </it>transposon
Abstract Background Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. Results We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. Conclusions This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.</p
Enhancer trapping in zebrafish using the Sleeping Beauty transposon
Background: Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. Results: We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADPribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. Conclusions: This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome. © 2004 Balciunas et al; licensee BioMed Central Ltd
Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation
Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation
<div><h3>Background</h3><p>Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.</p> <h3>Methodology/Principal Findings</h3><p>Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.</p> <h3>Conclusions/Significance</h3><p>We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity <em>in vitro</em>.</p> </div
Expression of LRRK2-GFP in U-2 OS cells via BacMam gene delivery.
<p>U-2 OS cells were transduced with 20% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. (A) GFP images were captured and representative images are shown. (B) Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody. The quantifications are from 3 independent experiments.</p
TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in SH-SY5Y cells.
<p>(A) SH-SY5Y cells were transduced with 25% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody was performed. The quantifications are from 3 independent experiments. (B) SH-SY5Y cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C and D1994A. Cells were plated onto a 384-well assay plate, lysed and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043580#pone-0043580-g002" target="_blank">Figure 2</a> legend. Emission ratios of 520 nm/495 nm are plotted against the amount of BacMam used for transduction. (C) Cells transduced with 25% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (B). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.</p
Top 16 hits of the Tocriscreen™ Mini library screen.
<p>The IC<sub>50</sub> values are the averaged numbers from three independent experiments for both cellular and biochemical TR-FRET assays and one representative data of two independent experiments for the cytotoxicity assay.</p
Tocriscreen™ mini library screening results.
<p>Average percent inhibition of library compounds was analyzed to produce a frequency distribution with binning equal to one percent increments. The frequency distribution was plotted and a gaussian curve fit to the distribution (curve statistics: mean 4.38% standard, deviation 10.53% and R2 value of 0.962).</p
The effects of hit compounds on LRRK2 phosphorylation at serines 910, 935, 955 and 973.
<p>Flp-In T-REx™ HEK293 GFP-LRRK2 G2019S cells (GS) or A2016T/G2019S (IRM) were induced and treated with indicated compounds at 20 µM. After cell lysis, GFP-LRRK2 was immunoprecipitated and the phosphorylation of LRRK2 was analyzed by immunoblotting. The quantifications are from 3 independent experiments.</p
TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in human neural stem cells.
<p>Human neural stem cells were transduced with 10% BacMam LRRK2-GFP G2019S or D1994A. Cells were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043580#pone-0043580-g002" target="_blank">Figure 2</a>. Emission ratios of 520 nm/495 nm are plotted against the concentration of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.</p