Autofluorescent Proteins with Excitation in the Optical ... - Tsien
Autofluorescent Proteins with Excitation in the Optical ... - Tsien
Autofluorescent Proteins with Excitation in the Optical ... - Tsien
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Animal Imag<strong>in</strong>g<br />
Transcription units conta<strong>in</strong><strong>in</strong>g mKate, dNeptune, or IFP1.1 followed by <strong>the</strong><br />
poliovirus IRES and a GFP cod<strong>in</strong>g sequence were constructed <strong>in</strong> pENTR1<br />
by standard methods, and <strong>the</strong>n recomb<strong>in</strong>ed <strong>in</strong>to pAd-CMV-DEST by <strong>the</strong><br />
Gateway system (Invitrogen). L<strong>in</strong>earized plasmid was used to transfect<br />
HEK293A cells for adenovirus production, followed by amplification and purification<br />
on FastTrap columns (Millipore) and elution <strong>in</strong>to HBSS + 10% glycerol.<br />
Serial dilutions onto HEK293A cells and GFP visualization were performed to<br />
assess titers. Injections, imag<strong>in</strong>g, and dissection were carried out <strong>in</strong> duplicate.<br />
Mice were anes<strong>the</strong>sized <strong>with</strong> ketam<strong>in</strong>e/xylaz<strong>in</strong>e, and <strong>the</strong>n <strong>in</strong>jected via <strong>the</strong> tail<br />
ve<strong>in</strong> <strong>with</strong> 50 mlof53 10 10 <strong>in</strong>fectious units per milliliter of purified adenoviruses.<br />
Five days later, mice were shaved on <strong>the</strong>ir ventral side and anes<strong>the</strong>sized <strong>with</strong><br />
ketam<strong>in</strong>e/xylaz<strong>in</strong>e. Images were acquired on a Maestro 1 animal fluorescence<br />
imag<strong>in</strong>g system (CRI) <strong>with</strong> <strong>in</strong>dicated excitation filters and a tunable liquid<br />
crystal emission filter of 40 nm bandwidth centered on <strong>the</strong> <strong>in</strong>dicated wavelengths.<br />
For quantification, off-animal dark counts were first subtracted, and<br />
<strong>the</strong>n mid-sagittal l<strong>in</strong>e <strong>in</strong>tensities were obta<strong>in</strong>ed <strong>in</strong> ImageJ <strong>with</strong> <strong>the</strong> Plot Profile<br />
function. Similar results were obta<strong>in</strong>ed <strong>in</strong> <strong>the</strong> duplicate samples for each condition.<br />
For <strong>the</strong> charts <strong>in</strong> Figure 3, l<strong>in</strong>e <strong>in</strong>tensities from duplicate conditions were<br />
averaged and background was def<strong>in</strong>ed as <strong>the</strong> mean mid-sagittal <strong>in</strong>tensity of<br />
un<strong>in</strong>fected liver, treatment-matched <strong>in</strong> terms of pre- or post-dissection condition.<br />
Def<strong>in</strong><strong>in</strong>g background as <strong>the</strong> mid-thoracic region of <strong>in</strong>fected animals gave<br />
similar results. For confocal imag<strong>in</strong>g, three randomly oriented freshly prepared<br />
liver slices were placed on a coverslip and imaged <strong>with</strong> a 403 water objective<br />
on a Zeiss LSM 5Live microscope. For each slice, fluorescence <strong>in</strong>tensities<br />
were measured from maximum <strong>in</strong>tensity projections of a ten section stack of<br />
total thickness of 40 mm. All animal procedures were performed accord<strong>in</strong>g<br />
to protocols approved by <strong>the</strong> University of California, San Diego, <strong>in</strong>stitutional<br />
animal care and use committee. For spectral unmix<strong>in</strong>g, <strong>the</strong> predicted Neptune<br />
distribution <strong>in</strong> <strong>the</strong> <strong>in</strong>frared image is removed based on <strong>the</strong> Neptune distribution<br />
<strong>in</strong> <strong>the</strong> far-red channel and <strong>the</strong> relative efficiencies <strong>in</strong> <strong>the</strong> two channels <strong>in</strong> detect<strong>in</strong>g<br />
Neptune, as calculated from <strong>the</strong> Neptune spectra.<br />
Crystallization and Ref<strong>in</strong>ement<br />
For crystallization, <strong>the</strong> His tag on <strong>the</strong> prote<strong>in</strong> was removed by TEV protease<br />
digestion. Crystals were grown by <strong>the</strong> sitt<strong>in</strong>g drop vapor diffusion method.<br />
One microliter of prote<strong>in</strong> at 10 mg/ml was mixed <strong>with</strong> 1 ml of 0.1 M HEPES<br />
(pH 7.0), 0.2 M CaCl2, and 20% PEG 6000 at 18 C. Crystals were cryoprotected<br />
<strong>with</strong> a mixture of 66.5% Paratone-N, 28.5% paraff<strong>in</strong> oil, and 5% glycerol<br />
(Sugahara and Kunishima, 2006) prior to immersion <strong>in</strong> liquid nitrogen.<br />
Data were collected at Beaml<strong>in</strong>e 8.3.1 at <strong>the</strong> Advanced Light Source, Lawrence<br />
Berkeley National Laboratory and processed <strong>with</strong> ELVES (Holton and<br />
Alber, 2004) and MOSFLM/SCALA (Evans, 2006; Leslie, 2006). The crystal<br />
structure was solved by molecular replacement us<strong>in</strong>g PHASER (McCoy<br />
et al., 2007) <strong>with</strong> mKate at pH 4.2 <strong>with</strong>out <strong>the</strong> chromophore (PDB 3BXA) as<br />
<strong>the</strong> search model. The structure was ref<strong>in</strong>ed <strong>with</strong> TLS parameters us<strong>in</strong>g<br />
PHENIX (Zwart et al., 2008), alternat<strong>in</strong>g <strong>with</strong> manual revision of <strong>the</strong> model<br />
us<strong>in</strong>g COOT (Emsley and Cowtan, 2004). No planarity restra<strong>in</strong>ts were applied<br />
to <strong>the</strong> chromophore dur<strong>in</strong>g ref<strong>in</strong>ement. Crystallographic data and ref<strong>in</strong>ement<br />
statistics are presented <strong>in</strong> Table 3. Structure validation was performed <strong>with</strong><br />
WHAT_CHECK (Hooft et al., 1996). Neptune and mKate (pH 7.0) (PDB file<br />
3BXB) were aligned and root mean square deviations were calculated us<strong>in</strong>g<br />
<strong>the</strong> align command <strong>in</strong> PyMol (http://www.pymol.org).<br />
ACCESSION NUMBERS<br />
Coord<strong>in</strong>ates have been deposited <strong>in</strong> <strong>the</strong> PDB <strong>with</strong> accession codes 3IP2.<br />
Sequences have been deposited <strong>in</strong> <strong>the</strong> EMBL Nucleotide Sequence Database<br />
<strong>with</strong> accession codes FN565568 (Neptune) and FN565569 (mNeptune), and<br />
<strong>in</strong> GenBank <strong>with</strong> accession codes GU189532 (Neptune) and GU189533<br />
(mNeptune).<br />
SUPPLEMENTAL DATA<br />
Supplemental Data <strong>in</strong>clude Supplemental Experimental Procedures, six<br />
figures, and four tables and can be found <strong>with</strong> this article onl<strong>in</strong>e at http://<br />
www.cell.com/chemistry-biology/supplemental/S1074-5521(09)00360-3.<br />
ACKNOWLEDGMENTS<br />
We thank Paul Ste<strong>in</strong>bach for assist<strong>in</strong>g <strong>with</strong> prote<strong>in</strong> photobleach<strong>in</strong>g and related<br />
analysis. We acknowledge <strong>the</strong> follow<strong>in</strong>g sources of support: <strong>the</strong> Jane Coff<strong>in</strong><br />
Childs Foundation and a Burroughs Wellcome Career Award for Medical<br />
Scientists (M.Z.L.), <strong>the</strong> University of California, San Diego, Chancellor’s Award<br />
for Undergraduate Research (M.R.M.), a Howard Hughes Medical Institute<br />
(HHMI) predoctoral fellowship (N.C.S.), a Canadian Institutes of Health<br />
Research postdoctoral fellowship (R.E.C.), <strong>the</strong> National Institutes of Health<br />
(NIH) Medical Scientist Tra<strong>in</strong><strong>in</strong>g Program and U.S. Army Breast Cancer<br />
Research Program (BCRP) grant W81XWH-05-01-0183 (T.A.A.), NIH grant<br />
R01 GM048958 (H-L.N., W.M., and T.A.), HHMI, NIH grants R01 GM086197<br />
and R37 NS027177, and BCRP grant W81XWH-05-01-0183 (R.Y.T.).<br />
Received: June 29, 2009<br />
Revised: October 5, 2009<br />
Accepted: October 12, 2009<br />
Published: November 24, 2009<br />
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