Finnzymes Oy

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News Article | December 23, 2015
Site: www.nature.com

C57BL/6 (B6, H-2b) and LP (H-2b) mice were obtained from Jackson Laboratory. B6 Lgr5-LacZ and B6 lgr5-gfp-ires-CreERT2 (Lgr5–GFP) mice were provided by H. Clevers1, 10. Mouse maintenance and procedures were done in accordance with the institutional protocol guideline of the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee. Mice were housed in micro-isolator cages, five per cage, in MSKCC pathogen-free facilities, and received standard chow and autoclaved sterile drinking water. To adjust for differences in weight and intestinal flora among other factors, identical mice were purchased from Jackson and then randomly distributed over different cages and groups by a non-biased technician who had no insight or information about the purpose or details of the experiment. The investigations assessing clinical outcome parameters were performed by non-biased technicians with no particular knowledge or information regarding the hypotheses of the experiments and no knowledge of the specifics of the individual groups. Isolation of intestinal crypts and the dissociation of cells for flow cytometry analysis were largely performed as previously described10. In brief, after euthanizing the mice with CO and collecting small and large intestines, the organs were opened longitudinally and washed with PBS. To dissociate the crypts, small intestine was incubated at 4 °C in EDTA (10 mM) for 15 min and then in EDTA (5 mM) for an additional 15 min. Large intestine was incubated in collagenase type 4 (Worthington) for 30 min at 37 °C to isolate the crypts. To isolate single cells from small and large intestine crypts, the pellet was further incubated in 1× TrypLE express (Gibco, Life Technologies) supplemented with 0.8 kU ml−1 DNase1 (Roche). For mouse organoids, depending on the experiments, 200–400 crypts per well were suspended in Matrigel composed of 25% advanced DMEM/F12 medium (Gibco) and 75% growth-factor-reduced Matrigel (Corning). After the Matrigel polymerized, complete ENR medium containing advanced DMEM/F12 (Sigma), 2 mM Glutamax (Invitrogen), 10 mM HEPES (Sigma), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (Sigma), 1 mM N-acetyl cysteine (Sigma), B27 supplement (Invitrogen), N2 supplement (Invitrogen), 50 ng ml−1 mouse EGF (Peprotech), 100 ng ml−1 mouse Noggin (Peprotech) and 10% human R-spondin-1-conditioned medium from R-spondin-1-transfected HEK 293T cells31 was added to small intestine crypt cultures10. For experiments evaluating organoid budding, the concentration of R-spondin-1 was lowered to 1.25–5%. For mouse large intestine, crypts were cultured in ‘WENR’ medium containing 50% WNT3a-conditioned medium in addition to the aforementioned proteins and 1% BSA (Sigma), and supplemented with SB202190 (10 μM, Sigma), ALK5 inhibitor A83-01 (500 nM, Tocris Bioscience) and nicotinamide (10 mM, Sigma). Media was replaced every 2–3 days. Along with medium changes, treatment wells received different concentrations of rmIL-22 (Genscript). We also tested the effects of F-652 (Generon Corporation). In some experiments, organoids from crypts were cultured in the presence of Stattic (Tocris Bioscience). For passaging of organoids, after 5–7 days of culture, organoids were passaged by mechanically disrupting with a seropipet and cold media to depolymerize the Matrigel and generate organoid fragments. After washing away the old Matrigel by spinning down at 600 r.p.m., organoid fragments were replated in liquid Matrigel. ISCs were isolated from Lgr5–GFP mice using a modified crypt isolation protocol with 20 min of 30 mM EDTA32, 33 followed by several strainer steps and a 5-min incubation with TrypLE and 0.8 kU ml−1 DNase1 under minute-to-minute vortexing to make a single-cell suspension. The Lgr5–GFPhigh cells were isolated by FACS. Approximately 5,000 ISCs were plated in 30 μl Matrigel and cultured in WENR media containing Rho-kinase/ROCK inhibitor Y-27632 (10 μM, Tocris Bioscience) and Jagged1 (1 μM, Anaspec). Starting from day 4, ISC were cultured without Wnt. For lymphocyte co-culture experiments, ILCs were isolated from the small intestine lamina propria. Washed small intestine fragments were incubated in EDTA/IEL solution (1× PBS with 5% FBS, 10 mM HEPES buffer, 1% penicillin/streptomycin (Corning), 1% l-glutamine (Gibco), 1 mM EDTA and 1 mM dithiothreitol (DTT)) in a 37 °C shaker for 15 min. The samples were strained (100 μM) and put in a Collagenase solution (RPMI 1640, 5% FCS, 10 mM HEPES, 1% penicillin/streptomycin, 1% glutamine, 1 mg ml−1 collagenase D (Roche) and 1 U ml−1 DNase1 (Roche) and incubated twice for 10 min in a 37 °C shaker. Afterwards, the samples were centrifuged at 1,500 r.p.m. for 5 min and washed with RPMI solution without enzymes. After several washes, the cell suspension was transferred into a 40% Percoll solution (in PBS), which is overlaid on an 80% Percoll solution. After spinning the interface containing the lamina propria, mononuclear cells was aspirated and washed in medium. The cell suspension was then stained with extracellular markers and Topro3 for viability. Topro3−CD45+CD11b−CD11c−CD90+ LPLs from B6 wild-type and Il22−/− mice and Topro3−CD45+CD3−RORγt+ ILC3s34 from Rorc(γt)-GFP+ mice (Jackson) were sorted for co-cultures with SI crypts. (For antibodies used, see Supplementary Table 1.) To activate and maintain LPLs and ILCs in culture, rmIL-2 (1,000 U ml−1), rmIL-15 (10 ng ml−1), rmIL-7 (50 ng ml−1) and rmIL-23 (50 ng ml−1) were added to the ENR medium in co-culture experiments. We have also performed co-cultures with addition of only rmIL-23 (50 ng ml−1) to ENR media. LPLs and SI crypts were cultured in Matrigel with a 7:1 LPL:crypt ratio; ILCs and crypts were cultured in Matrigel with a 25:1 ILC:crypt ratio. Co-cultures were compared to crypts cultured in ENR plus cytokines without LPLs or ILCs present. A neutralizing monoclonal antibody against IL-22 (8E11, Genentech)35 was used to abrogate IL-22-specific effects of ILCs. For specific experiments, organoids were cultured from fresh crypts obtained from specific genetically modified mice, such as the Stat1−/− mice (129S6/SvEv-Stat1tm1Rds, Taconic) and Stat3fl/fl mice (Jackson). Organoids from Stat3fl/fl mice that had been grown for 7 days were dissociated as single cells and incubated with adenoviral-Cre (University of Iowa) to cause the deletion of Stat3 from floxed organoid cells. Frozen passaged organoids from Lgr5DTR (Lgr5-DTR)25 mice were used to culture organoids in which Lgr5+ stem cells could be depleted with daily administration of diphtheria toxin (1 ng μl−1). For Paneth-cell-deficient organoid cultures, frozen crypts from Atoh1ΔIEC mice36 depleted of Paneth cells were used to culture organoids. As previously described36, Atoh1ΔIEC mice (and littermate controls) were given an intraperitoneal injection of tamoxifen (1 mg per mouse, Sigma, dissolved in corn oil) for 5 consecutive days to achieve deletion of ATOH1 from intestinal epithelium. Animals were euthanized on day 7 after the first injection, and intestinal crypts were isolated and frozen in 10% dimethylsulfoxide (DMSO) and 90% FBS. To investigate the effect of IL-22 on human small intestine, we generated human duodenal organoids from banked frozen organoids (>passage 7) that had been previously generated from biopsies obtained during duodenoscopy of three independent healthy human donors. All human donors had been investigated for coeliac disease, but turned out to have normal pathology. All provided written informed consent to participate in this study according to a protocol reviewed and approved by the review board of the UMC Utrecht, The Netherlands (protocol STEM study, METC 10-402/K). Human organoids were cultured in 10 μl Matrigel drops in expansion medium containing WENR with 10 nM SB202190, 500 nM A83-01 and 10 mM nicotinamide. For IL-22 stimulation experiments, rhIL-22 10 ng ml−1 (Genscript) was added daily. For the purpose of size measurements at day 6, organoids were passaged as single cells. Where applicable, organoid cultures were performed using conditioned media containing R-spondin-1 and WNT3a produced by stably transfected cell lines. R-spondin-1-transfected HEK293T cells31 were provided by C. Kuo. WNT3a-transfected HEK293T cells were provided by H. Clevers (patent WO2010090513A2). Cell lines were tested for mycoplasma and confirmed to be negative. For size evaluation, the surface area of organoid horizontal cross sections was measured. If all organoids in a well could not be measured, several random non-overlapping pictures were acquired from each well using a Zeiss Axio Observer Z1 inverted microscope and then analysed using MetaMorph or ImageJ software. Organoid perimeters for area measurements have been defined manually and by automated determination using the Analyze Particle function of ImageJ software, with investigator verification of the automated determinations, as automated measurements allowed for unbiased analyses of increased numbers of organoids. For automated size measurements, the threshold for organoid identification was set based on monochrome images. The sizes of the largest and smallest organoids in the reference well were measured manually, and their areas were used as the reference values for setting the minimal and maximal particle sizes. Organoids touching the edge of the images were excluded from the counting. After 5–7 days in culture, total organoid numbers per well were counted by light microscopy to evaluate growth efficiency. All organoid numbers were counted manually in this fashion except for the organoid counts presented in Extended Data Fig. 5b, which were counted using automated ImageJ analysis, as these organoids were too numerous to count manually. To compare organoid efficiency in different conditions, combining experiments with different organoid numbers, the percentage of organoids relative to the number of organoids in ENR-control (rmIL-22 0 ng ml−1) was calculated. The efficiency from sorted ISCs was presented as the percentage of cells forming organoids per number of seeded cells. BMT procedures were performed as previously described37. A minor histocompatibility antigen-mismatched BMT model (LP into B6; H-2b into H-2b) was used. Female B6 wild-type mice were typically used as recipients for transplantation at an age of 8–10 weeks. Recipient mice received 1,100 cGy of split-dosed lethal irradiation (550 cGy × 2) 3–4 h apart to reduce gastrointestinal toxicity. To obtain LP bone marrow cells from euthanized donor mice, the femurs and tibias were collected aseptically and the bone marrow canals washed out with sterile media. Bone marrow cells were depleted of T cells by incubation with anti-Thy 1.2 and low-TOX-M rabbit complement (Cedarlane Laboratories). The TCD bone marrow was analysed for purity by quantification of the remaining T cell contamination using flow cytometry. T cell contamination was usually about 0.2% of all leukocytes after a single round of complement depletion. LP donor T cells were prepared by collecting splenocytes aseptically from euthanized donor mice. T cells were purified using positive selection with CD5 magnetic Microbeads with the MACS system (Miltenyi Biotec). T cell purity was determined by flow cytometry, and was routinely approximately 90%. Recipients typically received 5 × 106 TCD bone marrow cells with or without 4 × 106 T cells per mouse via tail vein injection. Mice were monitored daily for survival and weekly for GVHD scores with an established clinical GVHD scoring system (including weight, posture, activity, fur ruffling and skin integrity) as previously described38. A clinical GVHD index with a maximum possible score of ten was then generated. Mice with a score of five or greater were considered moribund and euthanized by CO asphyxia. Recombinant mouse IL-22 was purchased from GenScript and reconstituted as described by the manufacturer to a concentration of 40 μg ml−1 in PBS. Mice were treated daily via i.p. injection with either 100 μl PBS or 100 μl PBS containing 4 μg rmIL-22. IL-22 administration was started on day 7 after BMT. This schedule was based on the results of rmIL-22 pharmacokinetics tested in untransplanted mice. For in vivo F-652 administration, starting from day 7 after BMT, mice were injected subcutaneously every other day for ten consecutive weeks with PBS or 100 μg kg−1 F-652. Mice were euthanized for organ analysis 21 days after BMT using CO asphyxiation. For histopathological analysis of GVHD, the small and large intestines were formalin-preserved, paraffin-embedded, sectioned and stained with haematoxylin and eosin. An expert in the field of GVHD pathology, blinded to allocation, assessed the sections for markers of GVHD histopathology. As described previously38, a semiquantitative score consisting of 19 different parameters associated with GVHD was calculated. For evaluation of stem-cell numbers, small intestines from Lgr5-LacZ recipient mice that were transplanted with LP bone marrow (and T cells where applicable) were collected. β-galactosidase (LacZ) staining was performed as previously described previously1. Washed 2.5-cm-sized small intestine fragments were incubated with an ice-cold fixative, consisting of 1% formaldehyde, 0.2% NP40 and 0.2% gluteraldehyde. After removing the fixative, organs were stained for the presence of LacZ according to manufacturer’s protocol (LacZ staining kit, Invivogen). The organs were then formalin-preserved, paraffin-embedded, sectioned and counterstained with Nuclear Fast Red (Vector Labs). Immunohistochemistry detection of REG3β was performed at the Molecular Cytology Core Facility of MSKCC using a Discovery XT processor (Ventana Medical Systems). Formalin-fixed tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems) and sections were blocked for 30 min with Background Buster solution (Innovex). Slides were incubated with anti-REG3β antibodies (R&D Systems, MAB5110; 1 μg ml−1) or isotype (5 μg ml−1) for 6 h, followed by a 60-min incubation with biotinylated goat anti-rat IgG (Vector Laboratories, PK-4004) at a 1:200 dilution. The detection was performed with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions. Slides were counterstained with haematoxylin (Ventana Medical Systems), and coverslips were added with Permount (Fisher Scientific). See Supplementary Table 1 for full description of antibodies used. Immunofluorescent staining was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using a Discovery XT processor (Ventana Medical Systems). Formalin-fixed tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), and antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 12 min. IL-22R antibodies (R&D Systems, MAB42; 0.1 μg ml−1) were applied and sections were incubated for 5 h followed by 60 min incubation with biotinylated goat anti-rat IgG (Vector Laboratories, PK-4004) at a 1:200 dilution. The detection was performed with streptavidin–horseradish peroxidase (HRP) D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen, T20932) prepared according to manufacturer’s instruction with predetermined dilutions. Next, lysozyme antibodies (DAKO, A099; 2 μg ml−1) were applied and sections were incubated for 6 h followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, PK6101) for 60 min. The detection was performed with streptavidin–HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 594 (Invitrogen, T20935) prepared according to manufacturer’s instruction with predetermined dilutions. Finally, GFP antibodies were applied and sections were incubated for 5 h followed by incubation with biotinylated goat anti-chicken IgG (Vector Laboratories, BA-9010) for 60 min. The detection was performed with streptavidin–HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 647 (Invitrogen, T20936) prepared according to manufacturer instruction with predetermined dilutions. Slides were counterstained with DAPI (Sigma Aldrich, D9542; 5 μg ml−1) for 10 min and coverslips were added with Mowiol. For immunofluorescent and other microscopic imaging, including LacZ and immunohistochemistry slides, contrast and white balance were set based on control slides for each experiment, and the same settings were used for all slides to maximize sharpness and contrast. See Supplementary Table 1 for full description of antibodies used. Spleen and small intestine were collected from euthanized BMT recipients, and organs were then homogenized and spun down. The supernatant was stored at −20 °C until use for cytokine analysis. The cytokine multiplex assays were performed on thawed samples with the mouse Th1/Th2/Th17/Th22 13plex (FlowCytomix Multiplex kit, eBioscience) and performed according to the manufacturer’s protocol. For in vivo experiments, lymphoid organs were collected from euthanized mice and processed into single cell suspension. Cells were stained with the appropriate mixture of antibodies. For intracellular analysis, an eBioscience Fixation/Permeabilization kit was used per the manufacturer’s protocol. After thorough washing, the cells were stained with intracellular and extracellular antibodies simultaneously. Fluorochrome-labelled antibodies were purchased from BD Pharmingen (CD4, CD8, CD24, CD25, CD45, α4β7 and P-STAT3 Y705, P-STAT1 Y701), eBioscience (FOXP3), R&D (IL-22R), and Invitrogen (GFP). DAPI and Fixable Live/Dead Cell Stain Kits (Invitrogen) were used for viability staining. Paneth cells were identified based on bright CD24 staining and side scatter granularity as described previously2. For flow cytometry of small intestine organoid cells, organoids were dissociated using TrypLE (37 °C). After vigorously pipetting through a p200 pipette causing mechanical disruption, the crypt suspension was washed with 10 ml of DMEM/F12 medium containing 10% FBS and 0.8 kU ml−1 DNase1 and passaged through a cell strainer. Where applicable, the cells were directly stained or first fixed (4% paraformaldehyde) and permeabilized (methanol) depending on the extracellular or intracellular location of the target protein. All stainings with live cells were performed in PBS without Mg2+ and Ca2+ with 0.5% BSA. For EdU incorporation experiments there was a 1 h pre-incubation of EdU in the ENR medium of the intact organoid cultures before dissociating the cells with TrypLE. Cells were stained using Click-it kits for imaging and flow cytometry (Life Technologies). For cell cycle analysis, single cell suspensions obtained from dissociated organoids were fixed and stained with Hoechst 33342 (Life Technologies), then assessed with flow cytometry for DNA content and ploidy. For intracellular pSTAT staining of organoids, organoids were mechanically disrupted into crypt fragments, stimulated for 20 min with 20 ng ml−1 IL-22 at 37 °C, and then fixed with 4% paraformaldehyde (10 min at 37 °C). To assess STAT activation in Lgr5+ cells, after freshly isolating crypts from Lgr5–GFP mice, single-cell suspensions including Y-27632 (10 μM) were stimulated with IL-22. After obtaining a single cell suspension of stimulated and fixed cells, the samples were filtered (40 μM) and permeabilized with ice-cold (−20 °C) methanol. Fixed and permeabilized cells were rehydrated with PBS and thoroughly washed with PBS before staining, then stained with anti-phospho-STAT3 and anti-phospho-STAT1, plus anti-GFP or cell surface markers, for 30 min at 4 °C. All flow cytometry was performed with an LSRII cytometer (BD Biosciences) using FACSDiva (BD Biosciences), and the data were analysed with FlowJo software (Treestar). See Supplementary Table 1 for full description of antibodies used. Western blot analysis was carried out on total protein extracts. Free-floating crypts isolated from small intestine were treated in DMEM supplemented with Y-27632 (10 ng ml−1, Tocris) and IL-22 (5 ng ml−1, 30 min). Vehicle (PBS) was added to control wells. Crypts were then lysed in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Sigma). After sonication, protein amount was determined using the bicinchoninic acid assay Kit (Pierce). Loading 30 μg per lane of lysate, proteins were separated using electrophoresis in a 10% polyacrylamide gel and transferred to nitrocellulose. Membranes were blocked for 1 h at room temperature with 1% Blot-Qualified BSA (Promega, W384A) and 1% non-fat milk (LabScientific, M0841) and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phospho-STAT1 (7649P), rabbit anti-phospho-STAT3 (9131S), rabbit anti-STAT1 (9172P) and rabbit anti-STAT3 (4904P), all from Cell Signaling. This was followed by incubation with the secondary antibody anti-rabbit HRP (7074P2) and visualization with the Pierce ECL Western Blotting Substrate (Thermo Scientific, 32106). Cell viability in organoids was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) test, based on the identification of metabolically active cells. The organoids were incubated with MTT (0.9 mg ml−1 final concentration, Sigma) for 2 h at 37 °C. Matrigel and cells containing intracellular reduction end product formazan were solubilized with acidic isopropanol (isopropanol with HCl) and the reduction end formazan production was evaluated by spectrophotometry using the Infinite M1000 pro plate reader (Tecan). For qPCR, segments of small intestine or isolated crypts were collected from euthanized mice and stored at −80 °C. Alternatively, RNA was isolated from organoids after in vitro culture. Extracted RNA was also stored at −80 °C. Reverse transcriptase PCR (RT–PCR) was performed with a QuantiTect Reverse Transcription Kit (QIAGEN) or a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). qPCR was performed on a Step-One Plus or QuantStudio 7 Flex System (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Specific primers were obtained from Applied Biosystems: Actb: Mm01205647_g1; Hprt: Mm00446968_m1; Reg3b: Mm00440616_g1; Reg3g: Mm00441127_m1; Wnt3: Mm00437336_m1; Egf: Mm00438696_m1; Rspo3: Mm00661105_m1; Axin2: Mm00443610_m1; Ctnnb1: Mm00483039_m1; Defa1: Mm02524428_g1; and Il22ra1: Mm01192943_m1. Other primers were obtained from PrimerBank: Gapdh (ID 6679937a1), Cdkn1a (also known as p21) (ID 6671726a1); Cdkn2d (also known as p19) (ID 31981844a1); Wnt3a (ID 7106447a1); Axin2 (ID 31982733a1); Hes1 (ID 6680205a1) Dll4 (ID 9506547a1) Dll1 (ID 6681197a1), for which cDNAs were amplified with SYBR master mix (Applied Biosystems) in QuantStudio 7 Flex System (Applied Biosystems). Relative amounts of mRNA were calculated by the comparative ΔC method with Actb, Hprt or Gapdh as house-keeping genes. For Il22ra1 qPCR on Lgr5+ cells, dissociated crypt cells from Lgr5–GFP mice were stained and isolated using the following monoclonal antibodies/parameters: EpCAM-1 (G8.8; BD Bioscience); CD45 (30F11; Life Technologies); CD31 (390; BioLegend), Ter119 (Ter119; BioLegend); GFP expression; dead cells were excluded using 7AAD. Cells were acquired on a BD ARIAIII and FACS-sorted. Cells were sorted directly into RA-1/TCEP (Macherey-Nagel) lysis buffer and stored at −80 °C until further analysis. RNA of haematopoietic cells (composite of dendritic cells, ILCs and B cells) was used as negative control. RNA was extracted using the NucleoSpin RNA XS kit (Machery Nagel) and cDNA was prepared with Ovation Pico and PicoSL WTA Systems V2 (NuGen). For qPCR, a Neviti Thermal Cycler (Applied Biosystems) and DyNAmo Flash SYBR Green qPCR kit (Finnzymes) were used, with the addition of MgCl to a final concentration of 4 mM. All reactions were done in duplicate and normalized to Gapdh. Relative expression was calculated by the cycling threshold (C ) method as 2−ΔCt. The primer sequences were as follows: Il22ra1: forward 5′-TCGGCTTGCTCTGTTATC-3′, reverse 5′-CCACTGAGGTCCAAGACA-3′. To explore the association of ISC gene signatures (GSE33948 and GSE23672)16 with STAT3-regulated genes, we performed GSEA in a mouse DSS colitis data set (GSE15955)12, comparing Stat3fl/fl;Villin-Cre− (wild type) and Stat3fl/fl;Villin-Cre+ (Stat3ΔIEC) mice with DSS colitis (GSEA2-2.2.0; http://www.broadinstitute.org/gsea)39, 40. A Paneth cell signature gene set was used as a negative control (DLL1+CD24hi, GSE39915)17. Nominal P values are shown. No statistical methods were used to predetermine sample size. To detect an effect size of >50% difference in means, with an assumed coefficient of variation of 30%, common in biological systems, we attempted to have at least five samples per group, particularly for in vivo studies. All experiments were repeated at least once. No mice were excluded from experiments. Experiments that were technical failures, such as experiments in vitro where cultures did not grow or experiments in vivo where transplanted control mice (bone marrow plus T cells) did not develop GVHD, were not included for analysis. Occasional individual mice that died post-transplant before analysis could not be included for tissue evaluation. All data are mean and s.e.m. for the various groups. Statistics are based on ‘n’ biological replicates. All tests performed are two sided. For the comparisons of two groups, a t-test or non-parametric test was performed. Adjustments for multiple comparisons were made. In most cases, non-parametric testing was performed if normal distribution could not be assumed. RT–qPCR reactions and ordinal outcome variables were tested non-parametrically. All analyses of statistical significance were calculated and displayed compared with the reference control group unless otherwise stated. There is large biological variation in organoid size. Statistical analyses of organoid sizes were thus based on all evaluable organoids (at least 25 organoids per group for all experiments). Statistical analyses of organoid numbers and efficiency were based on individual wells. To take into account intra-individual and intra-experimental variation as well, all in vitro experiments were performed at least twice with several wells per condition, and sample material coming from at least two different mice. Statistical analyses of stem-cell numbers (Lgr5-LacZ mice) in vivo were performed on several independent sections from multiple mice. Statistics were calculated and display graphs were generated using Graphpad Prism.


Trademark
Finnzymes Oy and Finnzymes Instruments Oy | Date: 2011-03-29

Laboratory apparatus and instruments, namely, thermal cyclers, microcentrifuges, polymerase chain reaction (PCR) plates, microplates for preparing and analyzing PCR samples, plate frames, tube and plate racks, test tubes and sample tubes. Medical diagnostic apparatus and instruments, namely, thermal cyclers and microcentrifuges for use in clinical diagnosis and analysis for human and animal diseases.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: KBBE-2007-1-3-01;KBBE-2007-1-1-02 | Award Amount: 3.94M | Year: 2008

Cattle-farming is one of the most important agricultural activities in the EU. This project will address issues of the health and welfare of cattle and the safety of cattle products, focussing on diseases that are on the increase in European cattle population and are aware of growing concern elsewhere. Two related diseases will be targeted, bovine tuberculosis (TB) and bovine para-tuberculosis (Para-TB or Johnes disease). The objective of the project is to use a combined functional and classical genomics and system biology approaches (system genetics) to investigate host-pathogen interactions and the host immune response to mycobacterium infection. The outcome will be 1) increased knowledge of macrophage function the application of this knowledge will be to develop tests to identify infected animals, and 2) the identification of genes that regulate the response of an individual to infection: information that could be applied in selective breeding programmes. Specifically the project will use functional, comparative genomics and in silico analysis to understand the genetic control of variation in the outcomes of disease challenge to develop molecular diagnosis tools to improve disease surveillance and to assist in selective improvement of breeding of cattle to control these diseases. Hence the project will impact directly on improved animal health. Healthy livestock are more productive and so the improvements achieved will contribute to improved efficiency and profitability of animal production and competitiveness of animal production and hence the sustainability of farming systems. The work will contribute both to improved animal health and welfare and also to the improved safety of animal products and to safeguarding human health. The market requirements will be assessed in order to lead the development of project outcomes to commercially viable products to ensure that the research is appropriately and efficiently exploited.


Patent
Finnzymes Oy | Date: 2011-10-04

Polymorphisms are present throughout an organisms genome, and understanding which alleles are present in a particular organisms genome can be advantageous. When probing the identity of these alleles, one must minimize incorrect readings due to inefficiencies in the system. In hydrolysis probe applications, these inefficiencies may be due to over-activity of an exonuclease functionality that excises nucleotides from probes that are only partially, complementary to a region of a target. The present invention provides a mixture that contains a plurality of polymerases including one that has a 53 exonuclease functionality and one that lacks or substantially lacks it, each in a sufficient relative amount and concentration to increase efficiencies of the system.


Patent
Finnzymes Oy | Date: 2011-09-09

The present invention relates to genetic engineering and especially to the use of DNA transposition complex of bacteriophage Mu. In particular, the invention provides a gene transfer system for eukaryotic cells, wherein in vitro assembled Mu transposition complexes are introduced into a target cell and subsequently transposition into a cellular nucleic acid occurs. The invention further provides a kit for producing insertional mutations into the genomes of eukaryotic cells. The kit can be used, e.g., to generate insertional mutant libraries.


Trademark
Finnzymes Oy | Date: 2011-09-20

Laboratory apparatus and instruments, namely, thermal cyclers, microcentrifuges, polymerase chain reaction (PCR) plates, microplates for preparing and analyzing PCR samples, plate frames, tube and plate racks, test tubes and sample tubes. Medical diagnostic apparatus and instruments, namely, thermal cyclers and microcentrifuges for use in clinical diagnosis and analysis for human and animal diseases.


Transposon nucleic acids comprising a transposon end sequence and a calibration sequence for DNA sequencing in the transposon end sequence. In one embodiment, the transposon end sequence is a Mu transposon end. A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with the calibration sequence providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates.


Patent
Finnzymes Oy | Date: 2010-10-04

The invention relates to a method of preparing a reaction mixture for Polymerase Chain Reaction (PCR) assay and a solution set for PCR. The method comprises providing a sample solution comprising a biological sample to be amplified in said PCR assay and first colorant providing the solution a first color, providing a reagent solution comprising at least one other substance required for performing said assay and second colorant providing the solution a second color different from the first color, and mixing the sample solution and the first reagent solution for providing a mixed solution to be subjected to the PCR process, the mixed solution having, due to said first and second colorants, a third color different from the first and second colors. The invention significantly aids in pipetting PCR assays.


A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with an engineered cleaveage site providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates. Transposon nucleic acids comprising a transposon end sequence and an engineered cleaveage site located in the sequence, e.g., in Mu transposon end sequence, are disclosed.


Patent
Finnzymes Oy | Date: 2011-04-01

The invention relates to a sample processing apparatus comprising a holder for a microtiter plate comprising a plurality of microwells, optical measurement unit for measuring optical responses of samples dosed to the microwells, and a computing unit configured to analyze the optical responses in order to detect dosing failures in said plurality of microwells, and if a dosing failure has been detected in one or more of the microwells, to communicate the existence of the dosing failure to a user of the apparatus through signaling means or to store data indicative of the dosing failure to data storage means for further use. In particular, the invention relates to detecting dosing failures in before, during and after a PCR process.

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