The Interdisciplinary Center for ... - University of Florida

The Interdisciplinary Center for ... - University of Florida

The Interdisciplinary Center for Biotechnology Research An Overview 2018 Pathology Research Infrastructure Talk April 11, 2018 Steven J. Madore, Ph.D. ICBR Associate Director for Science UFHCC Associate Director for Core Technologies Talk Outline The ICBR an introduction and historical overview Administrative Functions & Accessing ICBR Services Technologies & Expertise the ICBR Cores Introduction

Founded in 1987 by the Florida Legislature as a research support organization to develop biotechnology resources to support the UF research community. State of Florida and UF provide funding to support administration, scientific directors and technical personnel. Main facility located in Cancer & Genetics Research Complex with satellite locations in MacKnight Brain Institute and Microbiology and Cell Sciences. Mission is to enable, strengthen and energize all aspects of molecular life science research by providing scientific and technical instrumentation and expertise. Long term aim is to jump start research for technology transfer and accelerate molecular biology research success from concept through to data that advances science. Organized under the Vice President for Research, Dr. David Norton and led by Dr. Robert Ferl. Ease of use Cost effective Impactful

Your pursuit is what drives us. Organization Rob Ferl, Ph.D. ICBR Director Heather Voet Assistant Director Business Services Bioinformatics Next Generation

DNA Sequencing Monoclonal Antibodies Steven Madore, PhD Assoc Dir for Science Finance Manager Brad Barbazuk, PhD BI, Fac Dir, BI Cyberinfrastructure

Chris Vulpe, PhD CTC, Fac Dir Communications Andria Doty, PhD Cytometry, Sci Dir Human Resources Sixue Chen, PhD Proteomics, Sci Dir Paul Chipman, MA

EM Sci Dir Angel Sampson, PhD mAb Sci Dir Yanping Zhang, PhD GEG Sci Dir Electron Microscopy Flow Cytometry Business

Services CRISPR Technology New! Proteomics & Mass Spectrometry Gene Expression Easy access

Cost effective Overall ICBR Customer Satisfaction 1 (very dissatisfied) 2 3 4 5 (very satisfied) Not sure Frequency 1 4 9

25 35 1 % (N=75) 1.3% 5.3% 12.0% 33.3% 46.7% 1.3% What did the ICBR do really well? Angel Sampson was great about getting information to us at the start Came up with good suggestions and helped interpret the results

Communicate Communication (especially David Moraga) - Accommodate tight schedule. Communication when working through a project. Customer service, quality of training and data Easy requesting, timing EM core Expert advice on SEM use Fast service, constant updates, quality What could the ICBR do better? Better maintenance of instruments Bring back Sanger Sequencing Communication regarding technical issues Data analysis education Easier access/location to the college of medicine

Fewer password requirements to access scheduling calendars Talk Outline The ICBR an introduction and historical overview Administrative Functions & Accessing ICBR Services Technologies & Expertise the ICBR Cores Core Technologies Electron Microscopy Support researchers visualizing structures of microscopic samples full service imaging projects user training to facilitate solving research questions Major service categories include:

cryoEM, transmission and scanning electron microscopy confocal laser scanning and epifluorescence microscopy preparation of samples for microscopic analyses training users to operate microscopes and ancillary equipment Core Technologies Monoclonal Antibodies Development of monoclonal antibodies Immunization Fusion Cloning Monoclonal Ab production & purification Other services

Cell line cryostorage Monoclonal antibody cDNA sequencing of heavy and light chain variable regions cDNA Sequencing of Monoclonal Antibody Heavy and Light Chain Variable Regions Degenerate primers to conserved IgG HC regions and kappa chain LC primers were designed & used in RACE to amplify IgG HV & LV cDNAs from RNA prepared from hybridoma cell lines. HV LV RACE

from mRNA DNA Sequencing Alignment of predicted VH protein sequence from DNA sequencing cDNA Sequencing of Monoclonal Antibody Heavy and Light Chain Variable Regions Derived from CryoEM, ICBR Mavis Agbandje-McKenna, Ph.D. Director, UF Center for Structural Biology

Core Technologies Proteomics & Mass Spectrometry 1. Services and Technologies o o o o Protein fractionation Qualitative and quantitative mass spectrometry analysis de novo protein sequencing Post-translational modification analysis and accurate molecular weight determination o 2-D gel electrophoresis and Western blotting

o Protein enrichment and abundant protein depletion from biological fluids o Consultation, data processing and grant application support 2. Key equipment o Agilent 6495 Triple Quadrupole Mass Spectrometer coupled with the new Agilent 1290 UPLC Core Technologies Proteomics & Mass Spectrometry Protein mixture is enzymatically digested into peptides Peptide mix fractionated via HPLC

Analyzed by MS to generate spectrum Spectrum compared to databases of known peptides to identify proteins in original mixture M stands for mass and Z stands for charge number of ions. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labeling Labeling protein digests with isobaric tags allows many different biological samples to be mixed together in one analysis. Enables simultaneous identification of proteins including

relative and absolute quantitation. Low molecular weight reporter ions from the mass tags are generated by fragmentation in the mass spectrometer and their intensity is measured, the ratios of which determine the relative or absolute amount of protein in each sample Core Technologies Flow Cytometry 1. Services and Technologies Multi-parameter flow cytometry and live cell sorting

Training and Consultation; Courses and Workshops Confocal microscopy with live cell imaging Microparticle analysis using light scattering technology 2. Space and equipment 1400 sq. ft.- Cancer and Genetics Research Center, 682 sq. ft. -McKnight Brain Institute Key equipment: o o

o o Becton Dickenson LSR FORTESSA Flow Cytometer with 16 color 4 laser, 18 parameter analysis BD LSR II, CANTO II, Two - ACURI C6 flow cytometers, three - ARIA II/III SONY SH800 cell sorters 5-laser scanning Leica ST5 Confocal microscope with a live cell imaging incubation chamber NanoSight300 microparticle analyzer

Andria Doty, Ph.D. Scientific Director Flow Cytometry Using Fluorescently-labeled Antibodies Sorting Flow Cytometry Impact In vitro recognition of CD70 positive gliomas by human CD70 CAR T cells Cultured GBM cell lines were tested for the CD70 surface expression by FACS

CD70 overexpressed in GBM & associated with poor survival CD70 CAR T cells recognize expressing CD70 cells in vitro and induce antitumor response in pre-clinical models of GBM Targeting CD70 via CAR T immunotherapy could be employed to improve outcomes of GBM patients

Manipulation of tumor CD70 expression alters CAR T cells recognition. Human CAR T cells were cocultured with wild type GBM lines ( ) and GBM cells manipulated to overexpress CD70 ( ) or under-express CD70 via shRNA-mediated silencing ( ). Recognition was measured by IFN- release Core Technologies Gene Expression & Genotyping RNA analysis RNA qualitative/quantitative analysis qPCR and Droplet digital PCR mRNA splice variant analysis miRNA and non-coding RNA analysis RNASeq libraries

Whole transcriptome Sequence capture libraries (amplicon or exome) DNA genotyping Mouse tail DNA genotyping by PCR AFFYMETRIX AXIOM SNP genotyping Microsatellite analysis Human cell line authentication Core Technologies Bioinformatics Transcriptome analysis Metagenomics Genomic analysis of complex mixture of microorganisms Environmental samples, human gut microbiota Taxonomical resolution using whole genome vs 16S rRNA sequencing

Next-generation sequence analysis de novo genome assembly SVs, CNVs Statistical and functional analysis of microarray-based data Proteomics data analysis Core Technologies Next Generation DNA Sequencing 1. Services and Technologies Experimental design consultation Illumina short read DNA sequencing technology

PacBio long read DNA sequencing technology NGS library construction (whole genome, exome) RNASeq Epigenomics 2. Space and Equipment 3600 sq. ft. within the ICBR space in the Cancer and Genetics Research Complex (CGRC)

Key equipment includes: o o Illumina MiSeq (2), NextSeq, and HiSeq3000 DNA sequencers PacBio SEQUEL DNA sequencer Dr. David Moraga, Scientific Director ICBR NGS Core Advantages of Next Generation DNA Sequencing DNA Sequencing Faster and

Cheaper Illumi na PacBio SMRT Sequencing NGS Applications Whole genome sequencing De novo genome assembly Metagenomics Targeted sequencing Exome sequencing

Amplicon sequencing Variant & structural analysis 16S rRNA sequencing Transcriptomics (RNASeq) mRNA expression levels mRNA splice variant analysis Non coding RNAs (miRNAs, lncRNAs) Epigenetics DNA methylation Nucleic acid-Protein analysis Chromatin Immunoprecipitation (ChIP) RNA protein footprinting

Nucleosome footprinting Clinical diagnosis Tumor profiling Gene polymorphisms Fetal chromosome abnormalities Pharmacogenomics Sequencing Requires Shredding Chromosomal DNA and then Putting the ePieces Back Together Again! Electronic raw data

Compiled from raw data Whats next in NGS? New ICBR Core CRISPR Technology Co-managed by UFHCC and ICBR Led by Dr. Chris Vulpe, UFHCC Faculty Director Clustered Regularly Interspaced Short Palindromic Repeats Hallmark of a adaptive anti-phage immune system in bacteria first characterized in 1993 by Francisco Mojica, University of Alicante, Spain Simple enzymatic process modified to easily work in mammalian cells Gene editing in mammalian cells Generation of cell pools or clonal cell lines with targeted allele editing

CRISPR-based whole genome and targeted screening Gene Editing with CRISPR Cas9 gRNA Puromycin R QUESTIONS? Thank you!

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