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Cambridge, United Kingdom

Abcam plc is a global biotech company based in the Cambridge Science Park in Cambridge, UK, with offices in Cambridge , San Francisco, Hong Kong, Shanghai, and Tokyo. Wikipedia.


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— Cell culture protein surface coating is a procedure in which the cell culture surfaces are coated with proteins or extracellular matrix (ECM) components to enhance the adhesion and proliferation of the cells during in vitro isolation and cultivation. Proteins that are used in coating cells are collagen, fibronectin, vitronectin, laminin, and osteopontin, which are either animal derived, plant derived, synthetic, or human derived. Protein surface coating facilitates the growth of various types of cells such as epithelial, leukocytes, muscle cells, neurons, chinese hamster ovary (CHO) cell lines, fibroblasts, and neurons. Publisher's analysts forecast the global cell culture protein surface coating market to grow at a CAGR of 13.20% during the period 2017-2021. Covered in this report The report covers the present scenario and the growth prospects of the global cell culture protein surface coating market for 2017-2021. To calculate the market size, the report presents a detailed picture of the market by the way of study, synthesis, and summation of data from multiple sources. The market is divided into the following segments based on geography: - Americas - APAC - EMEA Publisher's report, Global Cell Culture Protein Surface Coating Market 2017-2021, has been prepared based on an in-depth market analysis with inputs from industry experts. The report covers the market landscape and its growth prospects over the coming years. The report also includes a discussion of the key vendors operating in this market. Get Sample of the Report at: http://www.reportsweb.com/inquiry&RW0001713445/sample . Other prominent vendors - Abcam - Agilent Technologies - AM-Pharma - BioLamina - BioMedTech Laboratories - Bio-Techne - BioTime - Caladrius Biosciences - Cedarlane Laboratories - Cell Guidance Systems - CellSystems Biotechnologie Vertrieb - Cellular Dynamics International - Cytoskeleton - Full Moon BioSystems - Greiner Bio-One - GlaxoSmithKline - Histocell - Japan Regenerative Medicine - KANGSTEM BIOTECH - Mesoblast - neuVitro - Orla Protein Technologies - Pall - PerkinElmer - PROGEN Biotechnik - PromoCell - RayBiotech - Sartorius Stedim Biotech - SouthernBiotech - Taiwan Bio Therapeutics - Takeda Pharmaceutical Company - Teva Pharmaceutical Industries - Trevigen - TWO CELLS - U.S. Stem Cell - Viogene Market driver - Government and research centers to promote research activities. - For a full, detailed list, view our report Market challenge - Ethical concerns over usage of animal-derived protein coating material. - For a full, detailed list, view our report Market trend - Increasing preference for 3D cell cultures over 2D cell cultures. - For a full, detailed list, view our report PART 01: Executive summary PART 02: Scope of the report PART 03: Market research methodology PART 04: Introduction PART 05: An overview: Cell culture surfaces PART 06: Market landscape PART 07: Market segmentation by product type PART 08: Geographical segmentation PART 09: Decision framework PART 10: Drivers and challenges PART 11: Market trends PART 12: Vendor landscape PART 13: Key vendor analysis PART 14: Appendix For more information, please visit http://www.reportsweb.com/global-cell-culture-protein-surface-coating-market-2017-2021


News Article | April 17, 2017
Site: globenewswire.com

Dublin, April 17, 2017 (GLOBE NEWSWIRE) -- Research and Markets has announced the addition of the "Quantitative Immunoassay Life Science Dashboard Series 3" report to their offering. Immunoassays are commonly used laboratory tests for identification and quantification of proteins. With a range of applications in diagnosis of cancer, immunology and disease research, immunoassays are widely used by scientists in academia and industry. The 2016 Quantitative Immunoassay Dashboard is the third in a series that characterizes the dynamic market for immunoassay products. This 2016 Dashboard provides a snapshot of the current market landscape that is compared with data from all of the previous Quantitative Immunoassay Dashboards , and was developed from responses to a 48-question survey completed by 592 scientists located in North America and Europe. This Dashboard reveals key market indicators for the Quantitative Immunoassay market as a whole as well as for the following techniques representing market sub-segments: - Colorimetric ELISA - Chemiluminescent ELISA - Fluorescent ELISA - Electrochemiluminescence immunoassays - Bead-based immunoassays - This report is focused on the use of Quantitative Immunoassay products in life science research market. The following suppliers were surveyed for this report: - Abcam - Affymetrix/eBioscience - BD Biosciences - Bio-Rad - GE Healthcare/Amersham - KPL/SeraCare - Luminex (direct) - Meso Scale Discovery - Perkin Elmer - R&D Systems/Biotechne - Roche Applied Science - Sigma/EMD Millipore - Thermo Fisher Scientific (including Life Technologies, Pierce, Invitrogen, Novex, Molecular Probes) Key Topics Covered: 1. Executive Summary 2. Dashboard "At A Glance" 3. Market Opportunity Matrix 4. Respondent Qualification 5. Demographics 6. Frequency of Performance: Life Science Techniques 7. Frequency of Use: Immunoassay Methods & Additional Protein Related Method 8. Co-Performance: Quantitative Immunoassay Methods & Life Science Techniques 9. Throughput, Growth Rates, Price & Monthly Spend 10. Market Size 11. Percentage of Spend with Suppliers of Immunoassay Products 12. Customer Satisfaction & Interest in Switching 13. Product Feature Influencing Purchase Decisions 14. Primary Focus/Application of Protein Analysis Research & Use of Protein Classes/Categories 15. Desired Changes to Immunoassay Products 16. Appendix I: Supporting Data 17. Appendix II: The Capabilities and Life Science Dashboards TM Available Companies Mentioned - Abcam - Affymetrix/eBioscience - BD Biosciences - Bio-Rad - GE Healthcare/Amersham - KPL/SeraCare - Luminex (direct) - Meso Scale Discovery - Perkin Elmer - R&D Systems/Biotechne - Roche Applied Science - Sigma/EMD Millipore - Thermo Fisher Scientific (including Life Technologies, Pierce, Invitrogen, Novex, Molecular Probes) For more information about this report visit http://www.researchandmarkets.com/research/ds2x4b/quantitative


Immunoassays are commonly used laboratory tests for identification and quantification of proteins. With a range of applications in diagnosis of cancer, immunology and disease research, immunoassays are widely used by scientists in academia and industry. The 2016 Quantitative Immunoassay Dashboard is the third in a series that characterizes the dynamic market for immunoassay products. This 2016 Dashboard provides a snapshot of the current market landscape that is compared with data from all of the previous Quantitative Immunoassay Dashboards , and was developed from responses to a 48-question survey completed by 592 scientists located in North America and Europe. This Dashboard reveals key market indicators for the Quantitative Immunoassay market as a whole as well as for the following techniques representing market sub-segments: - Colorimetric ELISA - Chemiluminescent ELISA - Fluorescent ELISA - Electrochemiluminescence immunoassays - Bead-based immunoassays - This report is focused on the use of Quantitative Immunoassay products in life science research market. The following suppliers were surveyed for this report: - Abcam - Affymetrix/eBioscience - BD Biosciences - Bio-Rad - GE Healthcare/Amersham - KPL/SeraCare - Luminex (direct) - Meso Scale Discovery - Perkin Elmer - R&D Systems/Biotechne - Roche Applied Science - Sigma/EMD Millipore - Thermo Fisher Scientific (including Life Technologies, Pierce, Invitrogen, Novex, Molecular Probes) Key Topics Covered: 1. Executive Summary 2. Dashboard "At A Glance" 3. Market Opportunity Matrix 4. Respondent Qualification 5. Demographics 6. Frequency of Performance: Life Science Techniques 7. Frequency of Use: Immunoassay Methods & Additional Protein Related Method 8. Co-Performance: Quantitative Immunoassay Methods & Life Science Techniques 9. Throughput, Growth Rates, Price & Monthly Spend 10. Market Size 11. Percentage of Spend with Suppliers of Immunoassay Products 12. Customer Satisfaction & Interest in Switching 13. Product Feature Influencing Purchase Decisions 14. Primary Focus/Application of Protein Analysis Research & Use of Protein Classes/Categories 15. Desired Changes to Immunoassay Products 16. Appendix I: Supporting Data 17. Appendix II: The Capabilities and Life Science Dashboards TM Available Companies Mentioned - Abcam - Affymetrix/eBioscience - BD Biosciences - Bio-Rad - GE Healthcare/Amersham - KPL/SeraCare - Luminex (direct) - Meso Scale Discovery - Perkin Elmer - R&D Systems/Biotechne - Roche Applied Science - Sigma/EMD Millipore - Thermo Fisher Scientific (including Life Technologies, Pierce, Invitrogen, Novex, Molecular Probes) For more information about this report visit http://www.researchandmarkets.com/research/w73jmf/quantitative To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/quantitative-immunoassay-life-science-dashboard-a-48-question-survey-completed-by-592-scientists---research-and-markets-300443417.html


No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Male heterozygous ythdf2+/− fish in the *AB background were custom made by ZGeneBio. TALEN mutagenesis was performed to mutate ythdf2 (Ensembl ENSDART00000127043) with L1 recognition sequence 5′-GGACCTGGCCAATCCCC-3′, R1 recognition sequence 5′-GGCACAGTAATGCCACC-3′, and spacer sequence 5′-TCCCAATTCAGGAATG-3′. Purchased fish were outcrossed to in-house wild-type *AB fish. Embryos were obtained from natural crosses, were raised under standard conditions, and were staged according to literature26. Embryos were reared at 28.5 °C and all experiments and observations were performed as close to this temperature as possible. Fish lines were maintained in accordance with AAALAC research guidelines, under a protocol approved by the University of Chicago IACUC (Institutional Animal Care & Use Committee). The open reading frame of zebrafish ythdf2 was purchased from Open Biosystems (clone 5601005) and subcloned into a pCS2+ vector using restriction enzyme sites of BamHI and XhoI. The resulting vector was linearized by HindIII and used as a template for ythdf2 probe preparation. Antisense digoxigenin (DIG) RNA probes were generated by in vitro transcription using standard reagents and methods. In situ hybridization protocol was followed essentially as previously reported27. All experiments were repeated at least once from biological samples. Control and ythdf2 morpholinos (5′-TGGCTGACATTTCTCACTCCCCGGT-3′) were obtained from Gene Tools (Oregon). 3 ng of either control or gene-specific morpholino was injected into *AB wild-type embryos at the one-cell stage. GFP and mCherry were subcloned into pCS2+ vectors and linearized by NotI. GFP-m6A, GFP-A, and mCherry-capped and polyadenylated mRNA was generated by in vitro transcription using mMessage mMachine SP6 kit (Thermo Fisher) and Poly(A) tailing kit (Thermo Fisher) according to the manufacturer’s protocol. Products were purified with the MEGAclear transcription clean-up kit (Thermo Fisher) and used for injections directly. For GFP-m6A, we spiked 6 nmol m6ATP into the 100 nmol ATP supplied in the transcription reaction, in order to ensure that less than 0.3% of GFP mRNAs are without m6A on average. (GFP mRNA is 942 nt; each mRNA has 1.89 m6A on average.) 35 pg of either GFP reporter mRNA and 10 pg of mCherry mRNA were injected together in 1.25 nl into embryos at the one-cell stage. ythdf2 mRNA containing the ythdf2 5′ UTR and a 3′ Flag tag, which was used to rescue the mutant phenotype and validate the knockdown efficiency of ythdf2 MO, was constructed in pCS2+ vector (forward primer: 5′-CGTACGGATCCTGTCTGATCTGCAGCTGTAG-3′; reverse primer: 5′-CGATGCTCGAGTTACTTGTCATCGTCGTCCTTGTAATCTATTCCAGATGGAGCAAGGC-3′) and prepared in the same way as mCherry mRNAs. Antibodies used in this study are listed below in the format of name (application; catalogue number; supplier): mouse anti-Flag HRP conjugate (Western; A5892; Sigma), rabbit anti-m6A (m6A-seq and m6A-CLIP-seq; 202003; Synaptic Systems), rabbit anti-histone H3 (IF; ab5176; Abcam), and anti-rabbit Alexa Fluor 488 (IF; ab150077; Abcam). All images were observed with a Leica MZFLIII microscope and captured with a Nikon D5000 digital camera using Camera Control Pro (Nikon) software. For fluorescent microscopy, standard ET-GFP and TXR LP filters (Leica) were used. For bright field imaging of live embryos, only saturation was adjusted and was adjusted identically for all images. For fluorescent imaging of live embryos, no image processing was performed. For fluorescent imaging of fixed embryos, contrast and exposure were adjusted for all to obtain the lowest amount of background while preserving the morphology of all visible nuclei. All experiments were repeated at least once from biological samples. To compare the total amount of DNA in wild-type and mutant embryos at different time points during the MZT, 10 embryos per time point per condition were dechorionated and pipetted into standard DNA lysis buffer. The number of embryos in each tube was counted twice to ensure uniformity. Proteinase K was added to 100 μg ml−1 and the embryos were incubated for 4 h at ~55 °C with occasional mixing. Proteinase K was inactivated by a 10-min incubation at 95 °C and the DNA was then phenol-chloroform-extracted, ethanol-precipitated, and resuspended in 100 μl Tris (pH 8.5) and 1 mM EDTA using standard procedures. Double-stranded DNA content was measured with NanoDrop. Three biological replicates (comprised of the offspring of three different fish mating pairs of the appropriate genotype) were measured for each time point for both the control and experimental samples. Biological replicates were averaged together to determine the average DNA amount per time point per genotype and to compute standard errors of the mean. All DNA values were normalized to that of wild-type embryos at 2.5 h.p.f. Embryos were collected into standard 2× protein sample buffer with added β-mercaptoethanol and protease inhibitors and immediately put on ice for a few minutes. The embryo mixtures were carefully but thoroughly pipetted up and down to dissolve and homogenize the embryos, and then samples were heated at 95 °C for 5 min and frozen at −80 °C. Before use, samples were again heated for 5 min and then centrifuged at 12,000 r.p.m. to remove debris. Supernatants were loaded into a 10-well, 1.5 mm Novex 4–20% Tris-Glycine Mini Protein Gel (Thermo Fisher) with 6 embryos per well. The gel was transferred onto a nitrocellulose membrane using iBlot2 gel transfer system (Thermo Fisher) set to P3 for 7 min with iBlot2 mini gel transfer stacks (Thermo Fisher). Membranes were blocked in 5% BSA, 0.05% Tween-20 in PBS for 1 h, and then incubated overnight at 4 °C with anti-Flag–HRP conjugate (Sigma) diluted 1:10,000 in 3% BSA. Proteins were visualized using the SuperSignal West Pico Luminol/Enhancer solution (Thermo Fisher) in FluorChem M system (ProteinSimple). mRNA isolation for LC-MS/MS: total RNA was isolated from zebrafish embryos with TRIzol reagent (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo). mRNA was extracted by removal of contaminating rRNA using RiboMinus Eukaryote Kit v2 (Thermo Fisher) for two rounds. Total RNA isolation for RT–qPCR: we followed the instruction of Direct-zol RNA MiniPrep kit (Zymo) with DNase I digestion step. Total RNA was eluted with RNase-free water and used for RT–qPCR directly. 100–200 ng of mRNA was digested by nuclease P1 (2 U) in 25 μl of buffer containing 10 mM of NH OAc (pH 5.3) at 42 °C for 2 h, followed by the addition of NH HCO (1 M, 3 μl, freshly made) and alkaline phosphatase (0.5 U). After an additional incubation at 37 °C for 2 h, the sample was diluted to 50 μl and filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μl of the solution was injected into LC-MS/MS. Nucleosides were separated by reverse-phase ultra-performance liquid chromatography on a C18 column with on-line mass spectrometry detection using an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside to base ion mass transitions of 282 to 150 (m6A), and 268 to 136 (A). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. The ratio of m6A to A was calculated on the basis of the calibrated concentrations9. Total RNA was isolated from fish embryos collected at different time points with TRIzol reagent and Direct-zol RNA MiniPrep kit. For each time point, ~200 embryos were collected to ensure RNA yield and that samples were representative. mRNA was further purified using RiboMinus Eukaryote Kit v2. RNA fragmentation was performed by sonication at 10 ng μl−1 in 100 μl RNase-free water using Bioruptor Pico (Diagenode) with 30 s on/off for 30 cycles. m6A-immunoprecipitation (IP) and library preparation were performed according to the previous protocol17. Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions. Additional high-throughput sequencing of zebrafish methylome was carried out using a modified m6A-seq method, which is similar to previously reported methods19, 20. Briefly, total RNA and mRNA were purified as previously described for m6A-seq. Purified mRNA (1 μg) was mixed with 2.5 μg of affinity purified anti-m6A polyclonal antibody (Synaptic Systems) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl (pH 7.4)) and incubated for 2 h at 4 °C. The mixture was subjected to UV-crosslinking in a clear flat-bottom 96-well plate (Nalgene) on ice at 254 nm with 0.15 J for 3 times. The mixture was then digested with 1 U μl−1 RNase T1 at 22 °C for 6 min followed by quenching on ice. Next, the mixture was immunoprecipitated by incubation with protein-A beads (Invitrogen) at 4 °C for 1 h. After extensive washing, the mixture was digested again with 10 U μl−1 RNase T1 at 22 °C for 6 min followed by quenching on ice. After additional washing and on-bead end-repair, the bound RNA fragments were eluted from the beads by proteinase K digestion twice at 55 °C for 20 and 10 min, respectively. The eluate was further purified using RNA clean and concentrator kit (Zymo Research). RNA was used for library generation with NEBNext multiplex small RNA library prep kit (NEB). Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions. Total RNA was isolated from wild-type and mutant fish embryos collected at different time points with TRIzol reagent and Direct-zol RNA MiniPrep kit. For each time points, ~20 embryos were collected to ensure RNA yield and that samples were representative. mRNA was further purified using RiboMinus Eukaryote Kit v2. RNA fragmentation was performed using Bioruptor Pico as described previously. Fragmented mRNA was used for library construction using TruSeq stranded mRNA library prep kit (Illumina) according to manufacturer’s protocol. Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions. All samples were sequenced by Illumina Hiseq 2000 with single-end 50-bp read length. The deep-sequencing data were mapped to zebrafish genome version 10 (GRCz10). (1) For m6A-seq, reads were aligned to the reference genome (danRer10) using Tophat v2.0.14 (ref. 28) with parameter -g 1–library-type = fr-firststrand. RefSeq Gene structure annotations were downloaded from UCSC Table Browser. The longest isoform was used if the gene had multiple isoforms. Aligned reads were extended to 150 bp (average fragments size) and converted from genome-based coordinates to isoform-based coordinates, in order to eliminate the interference from introns in peak calling. The peak-calling method was modified from published work18. To call m6A peaks, the longest isoform of each gene was scanned using a 100 bp sliding window with 10 bp step. To reduce bias from potential inaccurate gene structure annotation and the arbitrary usage of the longest isoform, windows with read counts less than 1 out of 20 of the top window in both m6A-IP and input sample were excluded. For each gene, the read counts in each window were normalized by the median count of all windows of that gene. A Fisher exact test was used to identify the differential windows between IP and input samples. The window was called as positive if the FDR < 0.01 and log (enrichment score) ≥ 1. Overlapping positive windows were merged. The following four numbers were calculated to obtain the enrichment score of each peak (or window): (a) reads count of the IP samples in the current peak or window, (b) median read counts of the IP sample in all 100 bp windows on the current mRNA, (c) reads count of the input sample in the current peak/window, and (d) median read counts of the input sample in all 100 bp windows on the current mRNA. The enrichment score of each window was calculated as (a × d)/(b × c). (2) For m6A-CLIP-seq, after removing the adaptor sequence, the reads were mapped to the reference genome (danRer10) using Bowtie2. Peak calling method was similar to the previous study19. Briefly, mutations were considered as signal and all mapped reads were treated as background. A Gaussian Kernel density estimation was used to identify the binding regions. The motif analysis was performed using HOMER29 to search motifs in each set of m6A peaks. The longest isoform of all genes was used as background. (3) For mRNA-seq, reads were mapped with Tophat and Cufflink (v2.2.1) was used to calculate the FPKM of each gene to represent their mRNA expression level30. (4) For fish gene group categorization, we used the input mRNA-seq data from m6A-seq. FPKM of all genes were first normalized to the highest value of five time points, with only genes with FPKM >1 analysed. Then Cluster3.0 (ref. 31) was used to divide all genes into six clusters, with the parameters: adjust data – normalize genes; k-means cluster – organize genes, 6 clusters, 100 number; k-means – Euclidean distance. The result clustered file with clustered number was merged with original FPKM values, imported and processed in R, and plotted in Excel. (5) For GO analysis, the list of target genes was first uploaded into DAVID32, 33 and analysed with functional annotation clustering. The resulting file was downloaded and extracted with GO terms and corresponding P values. The new list (contains GO terms with P < 0.01) was imported into REVIGO34 and visualized with the interactive graph, which was used as the final output figures. Methylated genes (at each time point) were defined as overlapped gene targets between m6A-seq and m6A-CLIP-seq. Ythdf2-regulated genes were defined as overlapped gene targets between the lists of the top 20% upregulated genes in both ythdf2 knockout and MO-injected samples. The most stringent Ythdf2 target genes at 4 h.p.f. (135) were defined in the main text, as overlapped genes of methylated genes at 4 h.p.f. (3,237) and Ythdf2-regulated genes at 4 h.p.f. (876). All the raw data and processed files have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and accessible under GSE79213. A summary of sequenced samples and processed FPKM data are included as Supplementary Data 2. One set of representative experiment results from at least two independent experiments were shown where applicable. Quantitative reverse-transcription PCR (RT–qPCR) was performed to assess the relative abundance of mRNA. All RNA templates used for RT–qPCR were pre-treated with on-column DNase I digestion in the purification step. RT–qPCR primers were designed to span exon-exon junctions to only detect mature mRNA. RT–qPCR was performed by using SuperScript III one-step RT–PCR system (Thermo Fisher) with 50–100 ng total RNA template. Actb1 was used as an internal control as it showed relative invariant expression during the studied time period according to pilot RT–qPCR data. P values were determined using two-sided Student’s t-test for two samples with equal variance. *P < 0.05; **P < 0.01; ***P < 0.001. The sequences of primers used in this study are listed below: actb1: forward 5′-CGAGCAGGAGATGGGAACC-3′, reverse 5′-CAACGGAAACGCTCATTGC-3′; buc: forward 5′-CAAGTTACTGGACCTCAGGATC-3′, reverse 5′-GGCAGTAGGTAAATTCGGTCTC-3′; zgc:162879: forward 5′-TCCTGAATGTCCGTGAATGG-3′, reverse 5′-CCCTCAGATCCACCTTGTTC-3′; mylipa: forward 5′-CCAAACCAGACAACCATCAAC-3′, reverse 5′-CACTCCACCCCATAATGCTC-3′; vps26a: forward 5′-AAATGACAGGAATAGGGCCG-3′, reverse 5′-CAGCCAGGAAAAGTCGGATAG-3′; tdrd1: forward 5′-TACTTCAACACCCGACACTG-3′, reverse 5′-TCACAAGCAGGAGAACCAAC-3′; setdb1a: forward 5′-CTTCTCAACCCAAAACACTGC-3′, reverse 5′-CTATCTGAAGAGACGGGTGAAAC-3′; mtus1a: forward 5′-TGGAGTATTACAAGGCTCAGTG-3′, reverse 5′-TTATGACCACAGCGACAGC-3′; GFP: forward 5′-TGACATTCTCACCACCGTGT-3′, reverse 5′-AGTCGTCCACACCCTTCATC-3′. High-throughput sequencing data that support the findings of this study have been deposited at GEO under the accession number GSE79213. All the other data generated or analysed during this study are included in the article and Supplementary Information.


News Article | February 15, 2017
Site: www.nature.com

C57BL/6N mice, ICR mice and Wistar rats were purchased from SLC Japan (Shizuoka, Japan). All animals were maintained under specific pathogen-free conditions. All animal experiments were approved by the Insititutional Animal Care and Use Committee, and performed in accordance with the guidelines of the University of Tokyo and the National Institute for Physiological Sciences. Diabetes was induced in 8-week-old C57BL/6N male mice by intravenous injection of 150 mg kg−1 of STZ. Mice with nonfasting blood glucose levels over 350 mg dl−1 1 week after STZ administration were used. Embryo culture and manipulation are described11. Rodent islets conventionally are isolated by collagenase perfusion of the pancreata through the common bile duct. However, the pancreata of Pdx1mu/mu chimaeric rats could not be perfused in this way because the pancreaticobiliary junction was maldeveloped in all (Extended Data Fig. 5). Therefore, we isolated islets by digestion of minced pancreata with collagenase. Pancreata removed from interspecific chimaera were inflated by interstitial injection of Gey’s balanced salt solution (GBSS; Sigma-Aldrich). GBSS-filled pancreata were minced using scissors. Small pieces of chopped pancreata were digested with collagenase XI (Sigma-Aldrich) to release islets from exocrine tissue. After 6–8 min incubation, islets were picked up using glass micropipettes and transplanted beneath the kidney capsule of 10-week-old male mice with STZ-induced diabetes, as previously described11. To prevent acute graft rejection, 0.5 mg per g (body weight) per day of tacrolimus, was injected intraperitoneally on the day of transplantation and on each of the following 4 days, in addition to an anti-inflammatory cocktail (all components, Affymetrix) containing anti-mouse interferon-γ mAb (rat IgGκ, 16-7312, clone R4-6A2), anti-mouse tumour necrosis factor-α mAb (rat IgG1κ, 16-7322, clone MP6-XT3) and anti-mouse IL-1β (hamster IgG, 16-7012, clone B122). The mRNA of TALENs (left and right) and rat Exo1 were generated by in vitro transcription. Linearized plasmids were transcribed from T7 promoter using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific) and resultant mRNAs were cleaned up by MEGAclear Kit (Thermo Fisher Scientific). 3 or 10 ng μl−1 of each mRNA was prepared by dilution in RNase- free-water and mixture of right TALEN, left TALEN and Exo1 were injected into the male pronuclei of zygotes by microinjection, as previously reported18. TALEN potential off-target sites were predicted by TALENoffer software. We chose 21 candidates (5 in exonic loci, 13 in intronic loci, 3 in intergenic loci) from TOP200 candidates19. We performed PCR amplification of genomic DNA from Pdx1+/muA, Pdx1+/muB and wild-type Wistar rats, subjecting the amplicons to Sanger sequencing. Genomic DNA was isolated from fluorescent-marker-negative cells isolated by FACS from chimaeric-rat blood samples. The TALEN target region of Pdx1 was amplified by PCR using the following primers: (forward) 5′-GCTGAGAGTCCGTGAGCTGCCCAG-3′ and (reverse) 5′-GGAACGCTTAAAGATCGTAGCAGC-3′). The PCR products were sequenced. Total RNA was isolated from duodenum of Pdx1muA/muB mice and reverse-transcribed by Superscript III reverse transcriptase (Thermo Fisher Scientific) with oligodT primer. Pdx1muA or Pdx1muB full-length cDNA were amplified by PCR using the following primers: (forward) 5′-GGCGCTGAGAGTCCGTGAGCTGC-3′ and (reverse) 5′-TTTTTTTTTTTTTTTGAAACCTCAAACAG-3′. Nonfasting blood glucose levels were determined (Medisafe-Mini glucometer; Terumo) weekly after islet transplantation. GTTs in overnight-fasted chimaera rats was conducted 0, 15, 30, 60 and 120 min after intraperitoneal injection of glucose (50% d-glucose solution, 2.5 g per kg body weight). Tail-vein blood was sampled by phlebotomy. Non-fasting serum mouse or rat c-peptide levels were analysed by enzyme-linked immunosorbent assay (ELISA) (mouse c-peptide ELISA kit, Shibayagi and Morinaga Institute of Biological Science; rat c-peptide ELISA kit, MERCODIA AB). Serum was isolated from 10-week-old Pdx1muA/muB + mPSCs chimaeras, C57BL/6N mice and Wistar rats. Serum was obtained from STZ-treated diabetic mice transplanted with mouseR islets 260 or 372 days after transplantation. SGE2 (EGFP-expressing mES cells) were derived from blastocysts generated from mating C57BL/6N female mice with C57BL/6N-Tg male mice (CAG-EGFP) (SLC Japan). mRHT (mES cells) were derived from blastocysts generated from mating male and female H2B-tdTomato knock-in mice with human histone H2B and tdTomato fusion gene in the mouse ROSA locus (T.K., unpublished data). Wlv3i-1 (rES cells) and GT3.2 (miPSCs) have been previously described11, 31. Maintenance of mPSCs and rPSCs has been previously described32, 33. Briefly, mPSCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 1,000 units per ml of mouse leukaemia inhibitory factor (all Thermo Fisher Scientific) and 1% l-glutamine-penicillin-streptomycin (Sigma-Aldrich). rPSCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts in N2B27 medium supplemented with 1 μM mitogen-activated protein kinase inhibitor PD0325901 and 3 μM glycogen synthase kinase inhibitor CHIR99021 (both Axon Groeningen). All PSC lines were authenticated by chimaera formation. These cell lines were not contaminated with mycoplasma. Isolated pancreata and islets were fixed in 4% paraformaldehyde in phosphate-buffered saline solution (PBS). Paraffin-embedded sections were incubated with blocking buffer (Active Motif) for 1 h at room temperature. The sections were incubated with primary antibodies, diluted in blocking buffer for 1 h at room temperature, and washed three times with PBS. They were then incubated with secondary antibodies for 1 h at room temperature. Primary antibodies used were guinea pig anti-insulin (Abcam; ab7842), rabbit anti-glucagon (Nichirei Bioscience, 422271), rabbit anti-somatostatin (Nichirei Bioscience 422651), rabbit anti-cytokeratin 19 (Abcam; ab52625, clone EP1580Y), mouse anti-amylase (SantaCruz; SC-46657, clone G-10) and goat anti-GFP (Abcam; ab6673), with Alexa-488-, Alexa-546-, and Alexa-633-conjugated secondary antibodies (Thermo Fisher Scientific). After antibody treatment, sections were mounted with Vectashield (Vector Laboratories), a mounting medium containing DAPI (Thermo Fisher Scientific) for nuclear counterstaining, and sections were observed under fluorescence microscopy. Three to five sections per slide were imaged and processed using Image J. For detection of lymphoid infiltration, DAB immunohistochemistry was performed with rabbit anti-CD3 (Abcam; ab5690) and rabbit anti-CD11b (Bioss Inc.; bs-1014R). Islets or small pieces of kidney that included transplanted islets were dispersed into single cells with collagenase type1A (Sigma-Aldrich). Dispersed cells stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (Thermo Fisher Scientific; A16201, clone 390) or allophycocyanin (APC)-conjugated anti-rat CD31 (Thermo Fisher Scientific; 50-0310-82, clone TLD-3A12) were subjected to FACS CantoII analysing (BD Biosciences). Data were collected for all of the dispersed cells and analysed. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Sample size was estimated on the basis of previous publications. Statistical significance was calculated by F-test and Student’s t-test (compare two groups) and the similarity to the Mendelian ratio was analysed by chi-square test (with Excel and Graphpad Prism software). P < 0.05 was considered to be statistically significant. Data are presented as mean ± s.d. Immunohistochemistry and flow-cytometry studies were repeated three times independently with similar results. All relevant data that are included with this study are available from corresponding auther upon reasonable request.


The invention provides a rabbit-derived immortal B-lymphocyte capable of fusion with a rabbit splenocyte to produce a hybrid cell that produces an antibody. The immortal B-lymphocyte does not detectably express endogenous immunoglobulin heavy chain and may contain, in certain embodiments, an altered immunoglobulin heavy chain-encoding gene. A hybridoma resulting from fusion between the subject immortal B-lymphocyte and a rabbit antibody-producing cell is provided, as is a method of using that hybridoma to produce an antibody. The subject invention finds use in a variety of different diagnostic, therapeutic and research applications.


A method for producing a library of engineered-antibody producing cells is provided. In certain cases, the method includes isolating nucleic acid sequences encoding IgH variable regions and IgL variable regions from a plurality of antibody producing cells, and introducing the nucleic acids into host cells to obtain cells that produce antibodies comprising non-naturally paired IgH and IgL variable chains.


Patent
Abcam | Date: 2014-02-10

In certain embodiments, the method may comprise: a) obtaining the antibody sequences from a population of B cells; b) grouping the antibody sequences to provide a plurality of groups of lineage-related antibodies; c) testing a single antibody from each of the groups in a bioassay and, after the first antibody has been identified, d) testing further antibodies that are in the same group as the first antibody in a second bioassay. In another embodiment, the method may comprise: a) testing a plurality of antibodies obtained from a first portion of an antibody producing organ of an animal; b) obtaining the sequence of a first identified antibody; c) obtaining from a second portion of said antibody producing organ the sequences of further antibodies that are related by lineage to said first antibody; and, c) testing the further antibodies in a second bioassay.


Patent
Abcam | Date: 2014-02-18

In certain embodiments, the method may comprise: a) obtaining the antibody sequences from a population of B cells; b) grouping the antibody sequences to provide a plurality of groups of lineage-related antibodies; c) testing a single antibody from each of the groups in a bioassay and, after the first antibody has been identified, d) testing further antibodies that are in the same group as the first antibody in a second bioassay. In another embodiment, the method may comprise: a) testing a plurality of antibodies obtained from a first portion of an antibody producing organ of an animal; b) obtaining the sequence of a first identified antibody; c) obtaining from a second portion of said antibody producing organ the sequences of further antibodies that are related by lineage to said first antibody; and, c) testing the further antibodies in a second bioassay.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 627.91K | Year: 2015

DESCRIPTION provided by applicant The most commonly used method for generating antibodies is through immunization of animals However this method is generally low throughput expensive time consuming and the antibodies generated are not always renewable Recombinant antibodies rAb like single chain variable fragments scFv have many attractive attributes compared to polyclonal antisera and monoclonal antibodies derived from hybridomas They are renewable through overexpression in the appropriate heterologous host they are easily stored and transferred as DNA and they can be genetically engineered as fusions to various enzymes fluorescent proteins and epitope tags While a number of approaches for generating recombinant affinity reagents exist the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable affinity reagents We believe that improvements to both gene synthesis technologies and the increased affordability of high throughput DNA sequencing can be leveraged to create antibody discovery pipelines based on synthetic biology that rival animal immune systems In this proposal we will build a high throughput pipeline for recombinant antibody development in as few as days The proposed platform takes advantage of pre designed diversity next generation sequence analysis and advanced molecular biology techniques to enable the rapid identification of specific antibodies Although our screening platform is being developed with single chain variable fragment antibodies scFvs the technology is applicable to both Fab and yeast display libraries High throughput conversion of the scFvs to full immunoglobulin G IgG will be integrated within the pipeline so that the antibodies can be directly validated in the desired final format In genomics it was thought that the $ genome would be the inflection point at which whole genome sequencing would become commonplace But even at $ per genome researcher uptake was phenomenal and new ways of using the NextGen sequencers like ChIP Seq RNA seq etc were invented In a similar fashion we believe that at $ and weeks researchers will begin to develop new applications where the cost of producing antibodies is no longer a relevant factor and speed becomes everything The ready availability of low cost high quality affinity reagents will potentially accelerate all aspects of basic science research provide diagnostics for disease biomarkers and serve as a proof of concept for therapy Like the $ genome we think antibody identification and production can eventually go to under $ and less than weeks At that point whole proteome analyses for many different organisms and disease states will be possible And new methods will arise PUBLIC HEALTH RELEVANCE At the moment it costs $ and $ to contract out production of rabbit polyclonal and monoclonal antibodies respectively Unfortunately these reagents take months to deliver may not be renewable cannot be engineered and their sequences are not known Recombinant antibodies rAb have many attractive attributes compared to polyclonal antisera and monoclonal antibodies derived from hybridomas They are renewable through overexpression in the appropriate host they are easily stored and transferred as DNA and they can be genetically engineered as fusions to various enzymes fluorescent proteins and tags While a number of approaches for generating recombinant affinity reagents exist the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable antibodies We believe that improvements to both gene synthesis technologies and the increased affordability of high throughput DNA sequencing can be leveraged to create antibody discovery pipelines based on synthetic biology that rival animal immune systems Here we present a platform for the rapid generation of recombinant monoclonal antibodies in as few as days at a cost comparable to that of polyclonals The platform takes advantage of pre designed library diversity that more closely mimics the natural diversity in human antibodies next generation sequence analysis to decode the enriched sequences and advanced molecular biology techniques to enable the rapid identification and production of recombinant antibodies In genomics it was thought that the $ genome would be the inflection point at which whole genome sequencing would become commonplace But even at $ per genome researcher uptake was phenomenal and new ways of using the NextGen sequencers like ChIP Seq RNA seq etc were invented In a similar fashion we do not know what the uptake and inflection points will be for antibodies At $ and weeks researchers may begin to develop new applications where the cost of producing antibodies is no longer a relevant factor and speed becomes everything The ready availability of low cost high quality affinity reagents will potentially accelerate all aspects of basic science research provide diagnostics for disease biomarkers and serve as a proof of concept for therapy Like the $ genome we think antibody identification and production can eventually go to under $ and less than weeks At that point whole proteome analyses for many different organisms and disease states will be possible And new methods will arise

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