Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner
1 Department of Cellular Biology and Anatomy, Georgia Health Sciences University, Augusta, GA 30912, USA
2 Vision Discovery Institute, Georgia Health Sciences University, Augusta, GA 30912, USA
3 Luminomics, Inc., Augusta, GA 30912, USA
4 Cancer Center, Georgia Health Sciences University, Augusta, GA 30912, USA
5 Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, D-85764 Germany
6 Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
7 Zoological Institute, Division of Cell Biology & Physiology, Braunschweig University of Technology, Braunschweig, 38106 Germany
8 Department of Neurobiology and Behavior, Stony Brook University, Stony Brook NY 11794, USA
BMC Biology 2012, 10:93 doi:10.1186/1741-7007-10-93Published: 30 November 2012
Additional file 1:
Diagram of transgene constructs. Schematics showing pertinent details of the transgenes tested and corresponding acronyms. Core elements include: cfos - minimal promoter ; KalTA4 - an optimized Gal4-VP16 fusion protein ; GI - rabbit beta-globin intron to promote mRNA stability ; pA - SV40 or bovine growth hormone polyadenylation sequences; 2xNRSE - a tandem repeat of a 21 bp consensus NRSE site , lox - loxP recombination sites to allow transgene cassette swapping ; UAS - 17 bp upstream activator sequence  specific for the Gal4 DNA binding domain (with indicated number of repeats, e.g., 5x or 14x); E1b - a basal promoter from carp beta-actin ; CREST1 - a 800-bp enhancer element characterized as a cranial motor neuron-specific element ; 2A - a porcine 2A viral peptide sequence  promoting equimolar expression of multicistronic messages ; nfsB - Escherichia coli gene encoding the prodrug converting bacterial enzyme nitroreductase (Ntr) which promotes chemically-induced cell ablation [52,53,88]; HE - a 365-bp promoter element from the zebrafish hatching enzyme 1a locus (he1a) that allows facile detection of UAS reporter lines in the absence of Gal4-VP16 driver elements (see Additional file 5). Fluorescent reporters included: M-YFP - a membrane-tagged (dual palmitoylation sequence from the Xenopus gap43 locus  'enhanced' yellow fluorescent protein (EYFP); M-tYFP - a membrane-tagged (same as above) monomeric 'tag' yellow fluorescent protein (tagYFP); mCherry - a monomeric red fluorescent protein .
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Additional file 2:
Enhancer trap comparisons ±NRSE. Confocal images of an additional 12 NRCK (left box) and 6 CK (right box) lines are shown in support of the phenotypic data summarized in Figure 1S. Each line is designated by a transgenic allele number (e.g., gmc601) and with the phenotypic characterization (e.g., Neural, Mixed, Non-Neural) provided in the lower right of each image set. Additional high resolution imaging data is available on line at .
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Additional file 3:
High-resolution imaging of branchiomotor neuron labeling. (A-E) Confocal images of 6-dpf NRC1CK-5xY2N transgenic line (Tg(2xNRSE-CREST1-cfos:KalTA4, 5xUAS-E1b:YFP-2A-nfsB)lmc003) showing specific labeling of branchiomotor neuron ganglia. When NRSE sites were placed upstream of CREST1-cfos, expression became restricted to cranial motor neuron subpopulations; the expression pattern originally characterized as CREST1-specified . (B, C) Motor ganglia expression included cranial nerve X (vagus, arrow in hindbrain region), VII (facial, down arrowhead), anterior and posterior V (trigeminal, up arrowhead); IV and III (trochlear and oculomotor, respectively, right arrowhead). (D, E) Unidentified descending spinal nerve.
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Additional file 4:
Early neuronal expression of NRSE Gal4 driver transgenes. Confocal images of 2-dpf triple transgenic line (Et(2xNRSE-cfos:KalTA4) gmc607; Tg(14xUAS:nfsB-mCherry)c264; Tg(elavl3:EGFP)knu3) showing typical early neural expression (arrows indicate double labeled neuronal cells) of NRCK lines (NRSE Gal4 drivers).
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Additional file 5:
Hatching enzyme promoter-based transgene 'tracer'. Stereoscope micrograph shows expression of he1a:YFP 'tracer' transgene in 1-dpf embryos. This element allows transgenic UAS reporter lines (e.g., Tg(loxP-5xUAS-E1b:gap43-YFP-loxP, he1a:gap43-YFP)gmc830, shown here) to be visually sorted from non-transgenic siblings (asterisks) at embryonic to early larval stages in the absence of Gal4 driver expression. The 365 bp he1a promoter is robustly active (arrow) from 1 to 3 dpf, after which expression rapidly fades. Inclusion of this element in UAS reporter lines has greatly simplified maintenance of our stocks.
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