News Article | March 16, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. The human iPS cell lines 201B7, 253G1, and 454E2 were obtained from the RIKEN Bio Resource Center (Tsukuba, Japan)21, 22. The 1231A3 and 1383D2 human iPS cells were provided by the Center for iPS Cell Research and Application, Kyoto University23. All cells were cultured in StemFit medium (Ajinomoto, Tokyo, Japan) on LN511E8-coated (0.5 μg cm−2) dishes23, 24. LN511E8, produced using cGMP-banked CHO-S cells (Life Technologies, Carlsbad, CA), was obtained from Nippi (Tokyo, Japan). In part, LN511E8 was produced using human 293-F cells as previously described12. The 201B7 and 454E2 human iPS cell lines were used in the in vitro experiments, while 201B7 and 1383D2 cells were used in the animal experiments; 253G1 and 1231A3 cells were used in the supplementary experiments, the results of which are reported in Extended Data Fig. 7. All of the experiments using recombinant DNA were approved by the Recombinant DNA Committees of Osaka University and were performed according to our institutional guidelines. The differentiation culture for human iPS cells was performed as indicated in Fig. 3a. First, human iPS cells were seeded on LN511E8-coated dishes at 350–700 cells cm−2, after which they were cultivated in StemFit medium for 8–12 days. The culture medium was then changed to DM (differentiation medium; GMEM (Life Technologies) supplemented with 10% knockout serum replacement (KSR; Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM non-essential amino acids (Life Technologies), 2 mM l-glutamine (Life Technologies), 1% penicillin-streptomycin solution (Life Technologies) and 55 μM 2-mercaptoethanol (Life Technologies) or monothioglycerol (Wako, Osaka, Japan))25. In some experiments, as indicated in the Results section, Noggin (R&D systems, Minneapolis, MN), LDN-193189 (Wako) or SB-431542 (Wako) were added for the first four days. BMP4 (R&D systems) was used in some early experiments at concentrations up to 0.125 nM. This had no discernible effect on SEAM formation, however, so its use was discontinued. After four weeks of culture in DM, the medium was changed to corneal differentiation medium (CDM; DM and Cnt-20 or Cnt-PR (w/o; EGF and FGF2) (1:1, CELLnTEC Advanced Cell Systems, Bern, Switzerland) containing 5 ng ml−1 FGF2 (Wako), 20 ng ml−1 KGF (Wako) 10 μM Y-27632 (Wako) and 1% penicillin-streptomycin solution). FGF2 in CDM was not essential for corneal epithelial induction. During CDM culture (around six to eight weeks of differentiation), non-epithelial cells were removed by manual pipetting under microscopy (Extended Data Fig. 2a, b). After pipetting, the medium was changed to fresh CDM. After four weeks of culture in CDM, the medium was changed to corneal epithelium maintenance medium (CEM; DMEM/F12 (2:1), Life Technologies) containing 2% B27 supplement (Life Technologies), 1% penicillin-streptomycin solution, 20 ng ml−1 KGF and 10 μM Y-27632 for two to seven weeks. To achieve retinal differentiation (Fig. 2c) after four weeks of differentiation the medium was directly changed to CEM. Isolated RPE cell colonies were cultivated in CEM on separate dishes coated with LN511E8. Phase-contrast microscopic observations were performed with an Axio-observer.Z1, D1 (Carl Zeiss, Jena, Germany) and an EVOS FL Auto (Life Technologies). Differentiated human iPS cells in CEM were dissociated using Accutase (Life Technologies), and resuspended in ice-cold KCM medium (DMEM without glutamine and Nutrient Mixture F-12 Ham (3:1, Life Technologies) supplemented with 5% FBS (Japan Bio Serum, Hiroshima, Japan), 0.4 μg ml−1 hydrocortisone succinate (Wako), 2 nM 3,3′,5-Triiodo-l-thyronine sodium salt (MP biomedicals, Santa Ana, CA), 1 nM cholera toxin (List Biological Laboratory, Campbell, CA), 2.25 μg ml−1 bovine transferrin HOLO form (Life Technologies), 2 mM l-glutamine, 0.5% insulin transferrin selenium solution (Life Technologies) and 1% penicillin-streptomycin solution). The harvested cells were filtered with a cell strainer (40 μm, BD Biosciences, San Diego, CA) and then stained with anti-SSEA-4 (MC813-70, Biolegend, San Diego, CA), TRA-1-60 (TRA-1-60-R, Biolegend) and CD104 (ITGB4; 58XB4, Biolegend) antibodies for 1 h on ice. After being washed twice with PBS, stained cells underwent cell sorting with a FACSAria II instrument (BD Biosciences). For intracellular protein staining, a BD Cytofix/Cytoperm (BD Biosciences) kit was used. In all of the experiments, cells were stained with non-specific isotype IgG or IgM as controls (Biolegend). The data were analysed using the BD FACSDiva Software (BD Biosciences) and the FlowJo software program (TreeStar, San Carlos, CA). Sorted human iPS cell-derived epithelial cells obtained from zone 3 of the SEAM (human iCECs) were seeded on LN511E8 coated (0.5 μg cm−2) cell culture inserts or temperature-responsive dishes (UpCell, CellSeed, Tokyo, Japan) without cell passaging, and were cultured in CEM until confluence26. To promote maturation, the epithelial cells were cultivated in CMM (corneal epithelium maturation medium; KCM medium containing 20 ng ml−1 KGF and 10 μM Y-27632) for an additional 3–14 days after CEM culture. The human iCECs cultivated on temperature-responsive dishes were released from their substrate by reducing the temperature to 20 °C. Total RNA was obtained from differentiated human iPS cells after specific culture periods, from human epidermal keratinocytes (EKs (foreskin), Life Technologies and TaKaRa Bio, Otsu, Japan), and from human corneal limbal epithelial cells (CECs) using the RNeasy total RNA kit or the QIAzol reagent (Qiagen, Valencia, CA). Reverse transcription was performed using the SuperScript III First-Strand Synthesis System for qRT–PCR (Life Technologies) according to the manufacturer’s protocol, and cDNA was used as a template for PCR. qRT–PCR was performed using the ABI Prism 7500 Fast Sequence Detection System (Life Technologies) in accordance with the manufacturer’s instructions. The TaqMan MGB used in the present study are shown in Supplementary Table 2. The thermocycling program was performed with an initial cycle at 95 °C for 20 s, followed by 45 cycles at 95 °C for 3 s and 60 °C for 30 s. Research grade human skin tissue sections were obtained from US Biomax Inc. (MD, USA) and human oral mucosal tissue was obtained from Science Care (Phoenix, AZ). The cells were fixed in 4% paraformaldehyde (PFA) or cold methanol, washed with Tris-buffered saline (TBS, TaKaRa Bio) three times for 10 min and incubated with TBS containing 5% donkey serum and 0.3% Triton X-100 for 1 h to block non-specific reactions. They were then incubated with the antibodies shown in Supplementary Table 3 at 4 °C overnight or at room temperature for 3 h. The cells were again washed twice with TBS for 10 min, and were incubated with a 1:200 dilution of Alexa Fluor 488-, 568-, 647-conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. Counterstaining was performed with Hoechst 33342 (Molecular Probes) before fluorescence microscopy (Axio Observer.D1, Carl Zeiss). Fabricated human iCEC sheets were fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque, Kyoto, Japan). After washing with distilled water, the human iCEC sheets were embedded in paraffin from which 3-μm-thick sections were cut. These were stained with haematoxylin and eosin following deparaffinization and hydration. The sections were observed with a NanoZoomer-XR C12000 (Hamamatsu Photonics, Hamamatsu, Japan), BZ-9000 (KEYENCE, Osaka, Japan) and an Axio Observer.D1. Differentiated human iPS cells (more than 12 weeks of differentiation) were fixed with 10% formaldehyde neutral buffer solution, after which PAS staining was performed with a PAS staining kit (MERCK KGaA, Darmstadt Germany) according to the manufacturer’s protocol. The sections were observed with an Axio Observer.D1. Epithelial cells were seeded onto MMC-treated NIH-3T3 feeder layers at a density of 3,000–20,000 cells per well. These were cultivated in CMM for 7–14 days. The colonies were fixed with 10% formaldehyde neutral buffer solution and then stained with rhodamine B (Wako). Colony formation was then assessed using a dissecting microscope and the colony-forming efficiency (CFE) was calculated. For the holoclone analysis, a single human iCEC colony derived from the SEAM was cultivated on 3T3-J2 (provided by H. Green, Harvard Medical School, Boston, MA) in CMM for 7–11 days was picked up under a dissecting microscope and dissociated by TrypLE Select (Life Technologies). The dissociated human iCECs were again seeded on a MMC-treated 3T3-J2 feeder layer and cultivated in CMM for 10–13 days. The colonies were scored under a microscope and classified as holoclones, paraclones or meloclones based on previously reported methods27. Human CECs were harvested from corneoscleral rims (Northwest Lions Eye Bank, Seattle, WA) as reported previously28. Human CECs and human oral keratinocytes (OKs; ScienCell, Carlsbad, CA) along with SEAM-derived human iCECs were cultivated on LN511E8 coated cell culture inserts in CEM until confluent. They were then cultivated in CMM. Human dermal fibroblasts (DFs; ScienCell) were cultivated in DMEM/F12 (2:1) containing 10% FBS. Total RNA was obtained from human iPS cells, iCECs, CECs, OKs, DFs, and six-week differentiated iPS cells (that is, OSE) using the QIAzol reagent. A microarray analysis using Sure Print G3 human 8x60K slides (Agilent technologies, Palo Alto, CA) was performed at Takara Bio. The data were analysed using the GeneSpring GX software program (Agilent technologies). Microarray data used in this study are deposited in Gene Expression Omnibus under accession number GSE73971. The cultivated epithelial cell sheets were fixed in 2.5% glutaraldehyde (Nacalai Tesque) at 4 °C overnight. Subsequently, the sheets were washed in buffer, dehydrated with ethanol and tert-butyl alcohol (Wako), and critical point dried (JFD-320, JEOL, Tokyo, Japan). After sputter-coating with platinum in an auto fine coater (JFCL-1600, JEOL), the samples were observed by scanning electron microscopy (JSM-6510LA, JEOL) at 5 kV. FACS-isolated human iCECs were cultivated on MMC-treated NIH-3T3 feeder layers in CMM up to 70–80% confluence. The human iCECs were harvested using TrypLE Select following the removal of feeder cells by manual pipetting. The total cell numbers were counted, after which the cells were passaged at a 1:8 ratio onto newly prepared feeder layers. These were cultivated in CMM until sub-confluence was reached again. The G-band karyotype analysis for human iCECs was performed at Nihon Gene Research Laboratories (Sendai, Japan). All animal experimentation was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the animal ethics committees of Osaka University. To examine embryonic mouse eyes, pregnant females (C57/BL6, E9.5–18.5) were acquired from SLC Japan (Shizuoka, Japan). For the transplantation experiments, Female New Zealand white rabbits (2.5–3.0 kg (approximately 12–14 weeks)) were obtained from Kitayama Labes (Nagano, Japan). Harvested human iCEC sheets were grafted onto rabbit corneas, in which a total epithelial limbal stem-cell deficiency had been created following a corneal and limbal lamellar keratectomy (Extended Data Fig. 8c–j). After surgery, 0.3% ofloxacin ointment (Santen Pharmaceutical, Osaka, Japan), 0.1% betamethasone phosphate eye drops (Shionogi Pharmaceutical, Osaka, Japan) and 0.1% sodium hyaluronate eye drops (Santen Pharmaceutical) were applied three to four times per day. Triamcinolone acetonide (8 mg; Bristol Myers Squibb, Tokyo, Japan) was also administered by subconjunctival injection. Tacrolimus (0.05 mg kg−1 per day, Astellas Pharma, Tokyo, Japan) and Mizoribine (4.0 mg kg−1 per day Sawai Pharmaceutical, Osaka, Japan) were systemically administered using an osmotic pump (DURECT, Cupertino, CA). The corneal barrier function following surgery was assessed by 0.5% fluorescein eye drop instillation at day 7 and day 14 after surgery and the fluorescein negative area was calculated using the AxioVision software program (Carl Zeiss). Throughout the healing period, the cornea was observed with a digital slit-lamp camera (SL-7F, TOPCON, Tokyo, Japan) and 3D OCT1000 MARK II (TOPCON) or CASIA SS-1000 (TOMEY, Nagoya, Japan) machines. If an infection was found or if unexpected weight loss occurred, animals were excluded from the analysis. The rabbits were euthanized by the intravenous administration of sodium pentobarbitone 14 days after transplantation, after which the eyes were immediately enucleated for the histological analyses. No blinding or randomization was conducted to allocate animals to each group. The data are expressed as means ± standard deviation (s.d.). The statistical analyses were performed using the Mann–Whitney rank sum test or Steel’s test. Bonferroni’s correction was applied to the data in animal experiments. All of the statistical analyses were performed using the JMP software program (SAS institute Inc., Cary, NC). No statistical methods were used to predetermine sample size. Comprehensive technical details can be found in Protocols Exchange, http://dx.doi.org/10.1038/protex.2016.009.
News Article | April 13, 2016
All non-transgenic, transgenic and control mice used in this study were derived from in house breeding colonies backcrossed > 12 generations onto C57/BL6 backgrounds. All mice used were young adult females between two and four months old at the time of spinal cord injury. All transgenic mice used have been previously well characterized or are the progeny of crossing well-characterized lines: (1) mGFAP-TK transgenic mice line 7.115, 16, 49; (2) mGFAP-Cre-STAT3-loxP mice generated by crossing STAT3-loxP mice with loxP sites flanking exon 22 of the STAT3 gene50 with mGFAP-Cre mice line 73.1217, 18; (3) loxP-STOP-loxP-DTR (diphtheria toxin receptor) mice21; (4) mGFAP-Cre-RiboTag mice generated by crossing mice with loxP-STOP-loxP-Rpl22-HA (RiboTag)26 with mGFAP-Cre mice line 73.1217, 18; (5) loxP-STOP-loxP-tdTomato reporter mice51. All mice were housed in a 12-h light/dark cycle in a specific-pathogen-free facility with controlled temperature and humidity and were allowed free access to food and water. All experiments were conducted according to protocols approved by the Animal Research Committee of the Office for Protection of Research Subjects at University of California, Los Angeles. All surgeries were performed under general anaesthesia with isoflurane in oxygen-enriched air using an operating microscope (Zeiss, Oberkochen, Germany), and rodent stereotaxic apparatus (David Kopf, Tujunga, CA). Laminectomy of a single vertebra was performed and severe crush spinal cord injuries (SCI) were made at the level of T10 using No. 5 Dumont forceps (Fine Science Tools, Foster City, CA) without spacers and with a tip width of 0.5 mm to completely compress the entire spinal cord laterally from both sides for 5 s16, 17, 18. For pre-conditioning lesions, sciatic nerves were transected and ligated one week before SCI. Hydrogels were injected stereotaxically into the centre of SCI lesions 0.6 mm below the surface at 0.2 μl per minute using glass micropipettes (ground to 50–100 μm tips) connected via high-pressure tubing (Kopf) to 10-μl syringes under control of microinfusion pumps, two days after SCI52. Tract tracing was performed by injection of biotinylated dextran amine 10,000 (BDA, Invitrogen) 10% wt/vol in sterile saline injected 4 × 0.4 μl into the left motor cerebral cortex 14 days before perfusion to visualize corticospinal tract (CST) axons, or choleratoxin B (CTB) (List Biological Laboratory, Campbell, CA) 1 μl of 1% wt/vol in sterile water injected into both sciatic nerves three days before perfusion to visualize ascending sensory tract (AST) axons33. AAV2/5-GfaABC1D-Cre (see below) was injected either 3 or 6 × 0.4 μl (1.29 × 1013 gc ml−1 in sterile saline) into and on either side of mature SCI lesions two weeks after SCI, or into uninjured spinal cord after T10 laminectomy. All animals received analgesic before wound closure and every 12 h for at least 48 h post-injury. Animals were randomly assigned numbers and evaluated thereafter blind to genotype and experimental condition. Adeno-associated virus 2/5 (AAV) vector with a minimal GFAP promoter (AAV2/5 GfaABC1D) was used to target Cre-recombinase expression selectively to astrocytes53, 54, 55. Diblock co-polypeptide hydrogel (DCH) K L was fabricated, tagged with blue fluorescent dye (AMCA-X) and loaded with growth factor and antibody cargoes as described38, 39, 52. Cargo molecules comprised: human recombinant NT3 and BDNF were gifts (Amgen, Thousand Oaks, CA, (NT3 Lot#2200F4; BDNF Lot#2142F5A) or were purchased from PeproTech (Rocky Hill, NJ; NT3 405-03, Lot#060762; BDNF 405-02 Lot#071161). Function blocking anti-CD29 mouse monoclonal antibody was purchased from BD Bioscience (San Diego, CA) as a custom order at 10.25 mg ml−1 (product #BP555003; lot#S03146). Freeze dried K L powder was reconstituted on to 3.0% or 3.5% wt/vol basis in sterile PBS without cargo or with combinations of NT3 (1.0 μg μl−1), BDNF (0.85 μg μl−1) and anti-CD29 (5 μg μl−1). DCH mixtures were prepared to have G′ (storage modulus at 1 Hz) between 75 and 100 Pascal (Pa), somewhat below that of mouse brain at 200 Pa (refs 38, 39). GCV (Cytovene-IV Hoffman LaRoche, Nutley, NJ), 25 mg kg−1 per day dissolved in sterile physiological saline was administered as single daily subcutaneous injections starting immediately after surgery and continued for the first 7 days after SCI. Bromodeoxyuridine (BrdU, Sigma), 100 mg kg−1 per day dissolved in saline plus 0.007 M NaOH, was administered as single daily intraperitoneal injections on days 2 through 7 after SCI. Diphtheria toxin A (DT, Sigma #DO564) 100 ng in 100 μl sterile saline was administered twice daily as intraperitoneal injections for ten days starting three weeks after injection of AAV2/5-GfaABC1D-Cre to loxP-DTR mice (which was 5 weeks after SCI) (see timeline in Extended Data Fig. 1d). Two days after SCI, all mice were evaluated in open field and mice exhibiting any hindlimb movements were not studied further. Mice that passed this pre-determined inclusion criterion were randomized into experimental groups for further treatments and were thereafter evaluated blind to their experimental condition. At 3, 7, 14 days and then weekly after SCI, hindlimb movements were scored using a simple six-point scale in which 0 is no movement and 5 is normal walking17. After terminal anaesthesia by barbiturate overdose mice were perfused transcardially with 10% formalin (Sigma). Spinal cords were removed, post-fixed overnight, and cryoprotected in buffered 30% sucrose for 48 h. Frozen sections (30 μm horizontal) were prepared using a cryostat microtome (Leica) and processed for immunofluorescence as described16, 17, 18. Primary antibodies were: rabbit anti-GFAP (1:1,000; Dako, Carpinteria, CA); rat anti-GFAP (1:1,000, Zymed Laboratories); goat anti-CTB (1:1,000, List Biology Lab); rabbit anti-5HT (1:2,000, Immunostar); goat anti-5HT (1:1,000, Immunostar); mouse anti-CSPG22 (1:100, Sigma); rabbit-anti haemagglutinin (HA) (1:500 Sigma); mouse-anti HA (1:3,000 Covance); sheep anti-BrdU (1:6,000, Maine Biotechnology Services, Portland, ME); rabbit anti-laminin (1:80, Sigma, Saint Louis, MO); guinea pig anti-NG2 (CSPG4) (E. G. Hughes and D. W. Bergles56, Baltimore, MA); goat anti-aggrecan (1:200, NOVUS); rabbit anti-brevican (1:300, NOVUS); mouse anti-neurocan (1:300, Milipore); mouse anti-phosphacan (1:500, Sigma); goat anti-versican (1:200, NOVUS); rabbit anti-neurglycan C (CSPG5) (1:200, NOVUS). Fluorescence secondary antibodies were conjugated to: Alexa 488 (green) or Alexa 350 (blue) (Molecular Probes), or to Cy3 (550, red) or Cy5 (649, far red) all from (Jackson Immunoresearch Laboratories). Mouse primary antibodies were visualized using the Mouse-on-Mouse detection kit (M.O.M., Vector). BDA tract-tracing was visualized with streptavidin-HRP plus TSB Fluorescein green or Tyr-Cy3 (Jackson Immunoresearch Laboratories). Nuclear stain: 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 2 ng ml−1; Molecular Probes). Sections were coverslipped using ProLong Gold anti-fade reagent (InVitrogen, Grand Island, NY). Sections were examined and photographed using deconvolution fluorescence microscopy and scanning confocal laser microscopy (Zeiss, Oberkochen, Germany). Axons labelled by tract tracing or immunohistochemistry were quantified using image analysis software (NeuroLucida, MicroBrightField, Williston, VT) operating a computer-driven microscope regulated in the x, y and z axes (Zeiss) by observers blind to experimental conditions. Using NeuroLucida, lines were drawn across horizontal spinal cord sections at SCI lesion centres and at regular distances on either side (Fig. 1a) and the number of axons intercepting lines was counted at 63× magnification under oil immersion by observers blind to experimental conditions. Similar lines were drawn and axons counted in intact axon tracts 3 mm proximal to SCI lesions and the numbers of axon intercepts in or near lesions were expressed as percentages of axons in the intact tracts in order to control for potential variations in tract-tracing efficacy or intensity of immunohistochemistry among animals. Two sections at the level of the CST or AST, and three sections through the middle of the cord for 5HT, were counted per mouse and expressed as total intercepts per location per mouse. To determine efficacy of axon transection after SCI, we examined labelling 3 mm distal to SCI lesion centres, with the intention of eliminating mice that had labelled axons at this location on grounds that these mice may have had incomplete lesions. However, all mice that had met the strict behavioural inclusion criterion of no hindlimb movements two days after severe crush SCI, exhibited no detectable axons 3 mm distal to SCI lesions regardless of treatment group. Sections stained for GFAP, CSPG or laminin were photographed using constant exposure settings. Single-channel immunofluorescence images were converted to black and white and thresholded (Fig. 1d and Extended Data Fig. 2b) and the amount of stained area measured in different tissue compartments using NIH ImageJ software. Areas are shown in graphs as mean values plus or minus standard error of the means (s.e.m.). Statistical evaluations of repeated measures were conducted by ANOVA with post hoc, independent pairwise analysis as per Newman-Keuls (Prism, GraphPad, San Diego, CA). Power calculations were performed using G*Power Software v18.104.22.168 (ref. 57). For quantification of histologically derived neuroanatomical outcomes such as numbers of axons or percentage of area stained for GFAP or CSPG, group sizes were used that were calculated to provide at least 80% power when using the following parameters: probability of type I error (α) = 0.05, a conservative effect size of 0.25, 2–8 treatment groups with multiple measurements obtained per replicate. Using Fig. 5j as an example, evaluation of n = 5 biological replicates (with multiple measurements per replicate) in each of 8 treatment groups provided greater than 88% power. For dot blot immunoassay of chondroitin sulfate proteoglycans (CSPG), spinal cord tissue blocks were lysed and homogenized in standard RIPA (radio-immunoprecipitation assay) buffer. LDS (lithium dodecyl sulfate) buffer (Life Technologies) was added to the post-mitochondrial supernatant and 2 μl containing 2 μg μl−1 protein was spotted onto a nitrocellulose membrane (Life Technologies), set to dry and incubated overnight with mouse anti-chondroitin sulfate antibody (CS56, 1:1000, Sigma Aldrich), an IgM-monoclonal antibody that detects glyco-moieties of all CSPGs22. CS56 immunoreactivity was detected on X-ray film with alkaline phosphatase-conjugated secondary antibody and chemiluminescent substrate (Life Technologies). Densitometry measurements of CS56 immunoreactivity were obtained using ImageJ software (NIH) and normalized to total protein (Poncau S) density58. Densities are shown in graphs as mean values plus or minus standard error of the means (s.e.m.). Two weeks after SCI, spinal cords of wild-type control (GFAP-RiboTag) and STAT3-CKO (GFAP-STAT3CKO-RiboTag) mice were rapidly dissected out of the spinal canal. The central 3 mm of the lower thoracic lesion including the lesion core and 1 mm rostral and caudal were then rapidly removed and snap frozen in liquid nitrogen. Haemagglutinin (HA) immunoprecipitation (HA-IP) of astrocyte ribosomes and ribosome-associated mRNA (ramRNA) was carried out as described26. The non-precipitated flow-through (FT) from each IP sample was collected for analysis of non-astrocyte total RNA. HA and FT samples underwent on-column DNA digestion using the RNase-Free Dnase Set (Qiagen) and RNA purified with the RNeasy Micro kit (Qiagen). Integrity of the eluted RNA was analysed by a 2100 Bioanalyzer (Agilent) using the RNA Pico chip, mean sample RIN = 8.0 ± 0.95. RNA concentration determined by RiboGreen RNA Assay kit (Life Technologies). cDNA was generated from 5 ng of IP or FT RNA using the Nugen Ovation 2 RNA-Seq Sytstem V2 kit (Nugen). 1 μg of cDNA was fragmented using the Covaris M220. Paired-end libraries for multiplex sequencing were generated from 300 ng of fragmented cDNA using the Apollo 324 automated library preparation system (Wafergen Biosystems) and purified with Agencourt AMPure XP beads (Beckman Coulter). All samples were analysed by an Illumina NextSeq 500 Sequencer (Illumina) using 75-bp paired-end sequencing. Reads were quality controlled using in-house scripts including picard-tools, mapped to the reference mm10 genome using STAR59, and counted using HT-seq60 with mm10 refSeq as reference, and genes were called differentially expressed using edgeR61. Individual gene expression levels in the Fig. 4e histogram are shown as mean FPKM (fragments per kilobase of transcript sequence per million mapped fragments). Additional details of differential expression analysis are described in the legends of Fig. 4 and Extended Data Figs 3 and 4. Raw and normalized data have been deposited in the NCBI Gene Expression Omnibus and are accessible through accession number GSE76097. To ensure the widespread distribution of these datasets, we have created a user-friendly website that enables searching for individual genes of interest https://astrocyte.rnaseq.sofroniewlab.neurobio.ucla.edu.
Fechter I.,Research Institute for Raw Materials |
Rath F.,Research Institute for Raw Materials |
Voetz M.,Biological Laboratory
Journal of the American Society of Brewing Chemists | Year: 2010
Due to the lack of molecular markers, assessment of the malting quality of newly bred barley lines is still limited to time-consuming and costintensive conventional analyses. In this study, we describe the development of a simple and robust PCR marker system for evaluating various germination-induced enzyme activities in spring barley, making it possible to predict overall malt quality without needing to malt the barley. Several feed and malting barleys were analyzed for their physical, chemical, and germinative properties. With respect to these properties, no systematic differences could be identified. During malting, however, the cultivars exhibited large differences in germination-induced enzyme activities, resulting in highly divergent malt quality. Gene expression studies using subtractive suppression hybridization and real-time reverse-transcription PCR resulted in the identification of a transcript that accumulated in concentrations that were up to 318-fold higher in the malting barleys than in most of the feed barleys. A simple and robust PCR marker system distinguishing between high and low enzyme activities could be set up and verified. With the help of this marker, for the first time it is possible to predict the potential malting quality of a barley cultivar without the necessity of running time-consuming and expensive malting and malt analyses. © 2010 American Society of Brewing Chemists, Inc.
Allen J.J.,Biological Laboratory |
Mathger L.M.,Biological Laboratory |
Barbosa A.,Biological Laboratory |
Barbosa A.,University of Porto |
And 6 more authors.
Proceedings of the Royal Society B: Biological Sciences | Year: 2010
Prey camouflage is an evolutionary response to predation pressure. Cephalopods have extensive camouflage capabilities and studying them can offer insight into effective camouflage design. Here, we examine whether cuttlefish, Sepia officinalis, show substrate or camouflage pattern preferences. In the first two experiments, cuttlefish were presented with a choice between different artificial substrates or between different natural substrates. First, the ability of cuttlefish to show substrate preference on artificial and natural substrates was established. Next, cuttlefish were offered substrates known to evoke three main camouflage body pattern types these animals show: Uniform or Mottle (function by background matching); or Disruptive. In a third experiment, cuttlefish were presented with conflicting visual cues on their left and right sides to assess their camouflage response. Given a choice between substrates they might encounter in nature, we found no strong substrate preference except when cuttlefish could bury themselves. Additionally, cuttlefish responded to conflicting visual cues with mixed body patterns in both the substrate preference and split substrate experiments. These results suggest that differences in energy costs for different camouflage body patterns may be minor and that pattern mixing and symmetry may play important roles in camouflage. © 2009 The Royal Society.
PubMed | Biological Laboratory
Type: Journal Article | Journal: The Journal of general physiology | Year: 2010
The ratio of electroosmotic to electrophoretic mobility of certain protein-coated surfaces is very close to 1.0, even in very dilute solutions of electrolytes.