ADVANCED TECHNOLOGY CORES CATALOG€¦ · I am pleased to present the BCM Advanced Technology Cores...

2017

ADVANCED TECHNOLOGY CORES CATALOG

ADVANCEDTECHNOLOGY

C O R E S

Dean P. Edwards, PhDExecutive Director

Dr. Edwards provides scientific oversight and guidance and establishes policies for governance and funding.

CORE LEADERSHIP

I am pleased to present the BCM Advanced Technology Cores catalog for 2017. This publication is designed to help you access the high-end instrumentation and specialized technologies you need for your research. The Advanced Technology Cores (ATC) at BCM expand the research capabilities of all researchers and essentially create unlimited research opportunities.

Each of the cores is staffed by a faculty level academic director, core directors and dedicated research technicians with highly specialized expertise in the technologies provided. A range

of research support services are provided such as access to shared instrumentation, analysis of research samples

provided by investigators and experiments with Core personnel performing specialized portions of the project. In addition to technical procedures, Cores

provide consultation on experimental design, data analysis and training.

This catalog provides an introduction to each of the Cores including services and major instrumentation/technology platforms, personnel, contact information and

examples of scientific research supported by core. For more information about any of the Cores, visit www.bcm.edu/research/corelabs.cfm.

On behalf of all the faculty and staff in the Cores, we look forward to working with you

to advance science across all areas at BCM.

— Adam Kuspa, PhDSenior Vice President for Research

ADVANCED TECHNOLOGY CORES

Javier G Villarreal, MBA Administrator

Mr. Villarreal administers financial and accounting policies and provides guidance for business operations.

Financial support to subsidize Core operations is provided by the following Institutional sources and

extra-mural grants.

INSTITUTIONAL SUPPORT

Dan L Duncan Comprehensive Cancer Center

Baylor College of Medicine Seed and Capital Funds

Office of Research: Advanced Technology Cores unit

GRANT SUPPORT

NCI P30 Cancer Center Support Grant (CCSG)

NIH P30 Digestive Disease Center (DDC)

NIH U54 Intellectual & Development Disabilities Research Center (IDDRC)

Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Awards

NEI P30 Instrumentation Module Center

NIH UM1 Consortium for large-scale production and phenotyping of knockout mice

ACKNOWLEDGEMENTS

Antibody-Based Proteomics . . . . 2

BioEngineering Core . . . . . . . . . . . . 3

Biostatistics and Informatics . . . . . . 4

Core for Advanced MRI (CAMRI) . . . . 6

Cell Based Assay Screening Service (C-BASS) . . . . . . . . . . . . . . . . . . . 8

Cytometry and Cell Sorting . . . . . . . . . . . .10

Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . 12

Genomic & RNA Profiling (GARP) . . . . . . . . . 14

Genetically Engineered Mouse (GEM). . . . . . . 16

Gene Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Human Tissue Aquisition and Patholgy (HTAP). . .20

Human Stem Cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Integrated Microscopy Core (IMC). . . . . . . . . . . . . . 23

Mass Spectrometry Proteomics . . . . . . . . . . . . . . . . . 24

Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

MHC Tetramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Mouse Embryonic Stem Cell Core . . . . . . . . . . . . . . . . . . . 29

Mouse Metabolism Core (MMC) . . . . . . . . . . . . . . . . . . . . . . 30

Mouse Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Optical Imaging and Vital Microscopy (OIVM) . . . . . . . . . . . . 34

Patient-Derived Xenograft and Advanced In Vivo Models Core . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Population Sciences Biorepository (PSB). . . . . . . . . . . . . . . . . . . . 38

Protein & Monoclonal Antibody Production. . . . . . . . . . . . . . . . . . . 40

RNA in Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Single Cell Genomics Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Small Animal MRI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Core Directory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

WHAT’S INSIDE

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CO R E LE AD E R S H I P

Shixia Huang, PhD

Academic Director

Associate Professor, Department of Molecular & Cellular Biology

713.798.8722

[email protected]

CO R E S U P P O R TE D R E S E ARCH

Proteomic profiles perturbed by FOXA1 overexpression are associated with multiple oncogenic pathways.FOXA1 mediated signaling pathways were dissected by reverse-phase protein arrays (RPPA) with 187 validated antibodies in Dox-inducible FOXA1-overexpressing ER+ cell models. The protein levels of ER and the products of its classical regulated genes (e.g., PR, BCL2, and MYC) were decreased in the MCF7L/FOXA1 +Dox cells. The GFR pathways of the focal adhesion, ERBB2, and insulin receptor were activated in both the MCF7L/FOXA1 +Dox and ZR75-1/FOXA1 +Dox cells (Fig. 1d and e). The tumour suppressor pathways (e.g., p53 and NOTCH in this analysis) were not perturbed by the FOXA1 overexpression. Less enhanced GFR signaling were found in the 600MPE/FOXA1 +Dox cells, possibly due to an endogenously hyper-activated MAPK pathway from a KRAS mutation in this line39 (Fig. 1f). Fu X, Jeselsohn R, Pereira R, Hollingsworth EF, Creighton CJ, Li F, Shea M, Nardone A, De Angelis C, Heiser LM, Anur P, Wang N, Grasso CS, Spellman PT,Griffith OL, Tsimelzon A, Gutierrez C, Huang S, Edwards DP, Trivedi MV, Rimawi MF, Lopez-Terrada D, Hilsenbeck SG, Gray JW, Brown M, Osborne CK, Schiff R. FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer. Proc Natl Acad Sci U S A. 2016: 113(43)

Figure 1. Heat maps of RPPA data representing differentially expressed proteins (one-way ANOVA, P < 0.05) in MCF7L/FOXA1 (a), ZR75-1/FOXA1 (b), and 600MPE/FOXA1 (c) cells upon Dox addition for 2 or 5 days. (d-f) FOXA1 augmentation-induced signaling perturbations in 14 cancer-related KEGG pathways were evaluated by averaging the expression levels of proteins available from RPPA (numbers in parentheses) within the same pathway, followed by subtraction of basal levels in -Dox cells. A paired one-sided t-test was applied and the P value was plotted as minus log10-transformed. The perturbations in pathways with P < 0.05 (P = 0.05 is marked by a dash-line) were statistically significant.

This Core provides customized services for protein profiling by antibody-based affinity protein array platforms. These platforms provide targeted quantitative assays both for validation and protein biomarker discovery, particularly for low abundance regulatory proteins and activation states of proteins with phosphorylated antibodies. Services provided include reverse phase protein arrays (RPPA) and Luminex bead technology for multiplex quantitative analyses of intracellular and extracellular signaling proteins.

MAJOR EQUIPMENT Bio-Plex 200 Luminex bead reader ( Bio-Rad) Luminex bead washer (Bio-Tek ELx405) Aushon 2470 protien arrayer Dako autostainer Link 4 Axon Array Scanner 4200AL and GenePix software (Molecular Devices) TissueLyzer II (Qiagen) NanoDrop 2000 Spectrophotometer (Thermo-Fisher) Molecular Devices Spectramax 340PC Plate Reader

SERVICES Reverse Phase Protein Array assays. High density microarrays spotted

with researchers’ protein lysates and probed with specific antibodies (>200 antibodies to proteins and phosphoproteins in multiple functional groups).

Luminex bead assays (Luminex xMAP technology) for highly sensitive quantitative measurement with very small protein lysate or serum samples.

Scanning of protein/antibody microarrays. Image analyses of protein/antibody microarrays. Data analysis Protein sample preparation. Consultation and experimental design.

ANTIBODY BASED PROTEOMICS

3WWW.BCM.EDU/CORELABS

The Core produced custom parts for several two-photon microscopes used to study neural information processing from the single cell to the network level.

Picture of an imaging setup used for studying motor-related activities in mice.

The goal of the Bioengineering Core is to provide investigators custom scientific instrumentation needed to conduct elegant experiments and ask truly cutting-edge research questions, and also to provide clinicians custom, one-of-a-kind, medical devices to create innovative solutions for health care. The core is staffed with experienced bioengineer and machinist who can work with investigators and clinicians to design complex devices, identify suitable off-the-shelf devices, manufacture custom parts, and integrate the apparatuses/instruments into the research work flow or clinical practices.

MAJOR EQUIPMENT Hermle 5-axis CNC (Computer Numerical Control) Milling machine center capable of cutting solid materials such as metal, plastics, and wood into parts with complex geometries up to a size of 24” x 18” x 18”.

Haas CNC Lathe – capable of machining custom cylindrical parts up to 14” diameter and 14” long.

Hardinge manual precision lathe. Bridgeport manual milling machine Vertical band saw and horizontal cutoff saw. Epilog Laser cutter – capable of cutting plastic, wood, or paper sheets up to 32” x 20” with 3/4” thickness and engraving plastic, leather, metal, and glass.

Stratasys 3D printer –capable of printing ABS plastics and supporting material up to a size of 8” x 8” x 6”.

Thorlabs optical workstation equipped with vibration isolation optical table, laser diode mount, laser controller, and power meter allowing tests of optical devices.

SERVICES Customized instrumentation design and manufacture. Customized electronics/optics design and manufacture. High precision mechanical manufacture. 3D design and printing. Laser cutting and engraving. Stockroom of fasteners and raw materials such as aluminum, stainless steel, and plastics.

Consultation for biomedical engineering projects.

BIOENGINEERING CORE (NEW 2017)

Fabrizio Gabbiani, PhD

Scientific Director

Professor

Department of Neuroscience

713.798.1849

[email protected]


I-Chih Tan, PhD

Engineer

Assistant Professor

Department of Neuroscience

713.798.9168

[email protected]


Schematics and pictures of a setup used to study visual information processing in insects.A: Single-facet stimulation setup for insect compound eyes and custom 2-photon microscope. B: sample image of the compound eye taken by a CMOS camera. C: sample image of the maximal z-projection of one identified neuron called the lobula giant move-ment stained with fluorescent dye.

Zhu Y and Gabbiani F; Fine and distributed subcellular retinotopy of excitatory inputs to the dendritic tree of a collision-detecting neuron, J Neurophysiol. 2016 Jun 1;115(6):3101-12

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The goal of this Core is to provide investigators with state of the art biostatistical, bioinformatic and biomedical informatic support for clinical, translational and basic science research.

MAJOR EQUIPMENTHighly-available cluster with >600 physical CPUs in a single compute node architecture with a 10 Gb Ethernet connection to extensible Tier 1 (ie, direct-attached, rapid I/O) NetApp data storage. Queues are managed with the PBS scheduler. The system is maintained by an expert HPC system administrator in a Tier III data center under standard governance structures.

SERVICES Biostatistics and Analytics Experimental design Assistance with design, & conduct of clinical trials Data analysis, including integrative bioinformatic analyses Statistical review for PRMS, DRC, etc. Multi-Omics Bioinformatics Data analysis for ‘omics core facilities’ Biomedical Informatics & Research IT OnCore®, Clinical Trials & Registry Data Management Acquire and Biobanking Data Management High Performance Computing (HPC) Software licensing for Oncomine™, Ingenuity®, SAS®, and SPSS®

Data Sharing Data sharing and research reproducibility statements Deposition of ‘omics-scale datasets Deposition of full-text articles in PubMed Central

BIOSTATISTICS AND INFORMATICS (REORGANIZED 2017)

CORE LEADERSHIP

Susan Hilsenbeck, PhD

Director

Professor, Department of Medicine, Smith Breast Center, Duncan Comprehensive Cancer Center

713.798.1632

[email protected]

Cristian Coarfa, PhD

Co-Director (Multi-Omics Bioinformatics)

Assistant Professor, Molecular & Cell Biology

713.738.7938

[email protected]

Hao Liu, PhD

Co-Director (Biostatistics)

Associate Professor, Duncan Cancer Center

713.798.5586

[email protected]

Chad Creighton, PhD

Co-Director (Cancer Bioinformatics)

Associate Professor, Duncan Cancer Center

713.798.2264

[email protected]

Neil McKenna, PhD

Co-Director (Data Sharing)

Associate Professor, Molecular and Cellular Biology

713.798.8568

[email protected]


In order to identify potential targets for molecularly targeted chemoprevention in cutaneous squamous cell carcinoma (cuSCC), we performed integrative cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. Functional pair analysis identified highly interconnected microRNA/mRNA regulatory networks that are conserved across species (see example of downregulated miRNA/upregulated mRNA), and we showed that cuSCC is molecularly related to carcinogen-driven SCCs of multiple sites (see CIRCOS plot). GSEA results for signatures of cancers profiled in the TCGA, with cancer types were ranked in descending order (clockwise). By this measure, cuSCC is most closely related to HNSC, LUSC, basal and HER2+ subtypes of breast cancer (BRCA) and ESCA SCC.

Chitsazzadeh V, Coarfa C, Drummond JA, Nguyen T, Joseph A, Chilukuri S, Charpiot E, Adelmann CH, Ching G, Nguyen TN, Nicholas C, Thomas VD, Migden M, MacFarlane D, Thompson E, Shen J, Takata Y, McNiece K, Polansky MA, Abbas HA, Rajapakshe K, Gower A, Spira A, Covington KR, Xiao W, Gunaratne P, Pickering C, Frederick M, Myers JN, Shen L, Yao H, Su X, Rapini RP, Wheeler DA, Hawk ET, Flores ER, Tsai KY: Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates. Nat Commun 7:12601, 2016, PMCID:PMC5013636, PMID:27574101

Multiplatform-based molecular subtypes of non-small-cell lung cancerWe integrated subtype classifications from five ‘omic data platforms to identify nine major lung cancer groups represented in TCGA (n=1023 cases). Molecular and clinical features that distinguish the lung cancer subtypes are shown.

Chen F, Zhang Y, Parra E, Rodriguez J, Behrens C, Akbani R, Lu Y, Kurie JM, Gibbons DL, Mills GB, Wistuba, II, Creighton CJ: Multiplatform-based molecular subtypes of non-small-cell lung cancer. Oncogene, 2016, PMID:27775076

Cross-species identification of genomic drivers

CORE SUPPORTED RESEARCH

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The Core for Advanced Magnetic Resonance Imaging (CAMRI) is a state-of-the-art resource for the Houston research community that makes possible advanced imaging studies of the function, physiology and anatomy of animals and humans, with special expertise in human blood-oxygen level dependent functional MRI (BOLD fMRI). Conveniently located in the heart of BCM main campus, the center houses three cutting edge MR imaging systems.

MAJOR EQUIPMENT Three Siemens 3 Telsa MRI Scanners. One Magnetom Trio and a PrismaFit system (the only Prisma scanner in Houston)

A wide variety of equipment for functional brain imaging studies, including sensory stimulation devices, response buttons, eye trackers, and MR-compatible transcranial magnetic stimulation (TMS).

Weekly journal club and seminar series, details on our Wiki at http://openwetware.org/wiki/CAMRI

Flywheel scientific data management system to make data easily accessible and shareable.

SEQUENCES Functional MRI (fMRI), including multiband acceleration Diffusion tensor imaging (DTI) Single and multi-voxel magnetic resonance spectroscopy (MRS)

Arterial spin labeling (ASL), both pulsed and continuous High-resolution structural imaging: FLASH, TSE, FLAIR, etc.

SERVICES Imaging technologist available to assist in data collection Physicist Support: Includes consultation and protocol development time, data management, and general collaborations on MRI projects

Pilot Time Program: Principal Investigators may apply for scanner time in order to collect preliminary data for grant proposals

Operator training available to enable safe use of MRI equipment by new users

Access to the instruments for fully trained users is available 24/7, facilitating subject recruitment and retention

CORE FOR ADVANCED MRI (CAMRI)

Michael S. Beauchamp, PhD Academic Director

Associate Professor, Department of Neurosurgery and Neuroscience

713.798.3175

[email protected]

CORE LEADERSHIP

Krista Runge, MS, RT(R)(MR)

Operations Director

713.798.3046

[email protected]



Work by Dr. David Ress focusses on the basic mechanisms of the BOLD signal, supported by R01 NS095933, “Measurements And Modeling Of The Hemodynamic Response Function In Human Cerebral Cortex”. Working with Assistant Professor Jung Hwan Kim, Dr. Ress has proposed a new model, the arterial impulse model, to explain the complex sequence of events linking changes in neuronal metabolic demand to MRI signal differences.

Figure from “Arterial impulse model for the BOLD response to brief neural activation.” Kim JH, Ress D. Neuroimage. 2016 Jan 1;124(Pt A):394-408. doi: 10.1016/j.neuroimage.2015.08.068.

Work by Dr. Michael Beauchamp examines the application of fMRI to a key human ability, the abil-ity to communicate face-to-face using speech. Speech consists of both auditory information from the talker’s voice and visual information from the talker’s face. Working with MD/PhD student Lin Zhu, Dr. Beauchamp has discovered a new organizing brain principle underlying multisensory speech perception. Using CAMRI’s trio scanner, Lin found that the natural statistics of human speech, in which voices co-occur with mouth movements, are reflected in the neural architecture of the superior temporal sulcus: different subregions prefer visually-presented faces containing either a moving mouth or moving eyes, but only mouth-preferring subregions respond strongly to voices. This discovery may help treat stroke patients who have lost the ability to understand speech and children who have language learning deficits.

Figure adapted from Zhu LL, Beauchamp MS. Mouth and Voice: A relationship between visual and auditory stimulus selectivity in the human superior temporal sulcus. Journal of Neuroscience (under review).

MRI can be used to measure brain activity using the blood oxygen level dependent (BOLD) signal.

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C-BASS strives to provide cutting-edge technologies and the latest genomic tools to BCM researchers in their cell-based functional genomics studies, and to aid their investigation of individual gene function, pathway identification, and large-scale genome-wide screens. The cell-based services we offer are built upon two interconnected and complementary technology platforms: RNAi-based functional genomics and CRISPR/Cas9-based genome editing. Services include generating knockout and knock-in cell lines using CRISPR/Cas9, providing cDNA and shRNA vectors individually or as custom libraries, and consultation and expert advice on genome-wide or subgenome targeted genetic screens. Through education and on-going improvement and optimization, we aim to enable BCM researchers to carry out drug discovery screens using a variety of platforms available in the core.

MAJOR RESOURCES The lentivirus-based shRNA library that targets the human genome, in both arrayed and pooled format

The lentivirus-based shRNA library that targets the mouse genome, in arrayed format A human cDNA library in a Gateway® compatible vector A mouse cDNA library A vector collection for CRISPR/Cas9-mediated genome editing

SERVICES

RNAi/cDNA vector resources Individual vectors Pre-assembled shRNA sublibraries (e.g., kinase, transcription factors, etc.) Custom sublibraries (gene collection designed by investigator) Whole-genome shRNA/cDNA collection (human and mouse) Lentiviral production and infection (individual or 96-well format)

CRISPR/Cas9 Consultation and experimental design for genome editing Vector design and construction Cell line generation and validation

CELL BASED ASSAY SCREENING SERVICE (C-BASS)

Trey Westbrook, PhD Academic Director

Associate Professor, Department of Biochemistry and Molecular Biology

713.798.5364

[email protected]

Dan Liu, PhD

Core Director


713.798.8032

[email protected]

CORE LEADERSHIP


In mammals, DNA methylation is essential for protecting repetitive sequences (including telomeres) from aberrant transcription, translocation, and homologous recombination. However, DNA hypomethylation occurs during specific developmental stages (e.g. preimplantation embryos) and in certain cell types (e.g., primordial germ cells). The absence of dysregulated repetitive elements in these cells suggests the existence of alternative mechanisms that prevent genome instability triggered by DNA hypomethylation. The researchers investigated the role of the chromatin factors Daxx and Atrx using genome-wide binding and transcriptome analysis, and found the two proteins to have distinct chromatin-binding profiles and co-enrichment at tandem repetitive elements. Further mechanistic studies were carried out using RNAi and CRISPR/Cas9 in wildtype and mutant mouse ES cells, and reveal Daxx and Atrx as new players that safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

He Q, Kim H, Huang R, Lu W, Tang M, Shi F, Yang D, Zhang X, Huang J, Liu D, Songyang Z. The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation. Cell Stem Cell 2015. 17(3):273-86.


Loss-of-function studies using RNAi and CRISPR/Cas9 reveal new functions for chromatin factors Daxx and Atrx

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CYTOMETRY AND CELL SORTINGFlow cytometry is an integral part of the research of BCM faculty across all disciplines. The technology continues to develop at a rapid pace driven by advances in fluorescent reagents, computational capacity and optics. The mission of the Core is to provide state-of-the-art instrumentation and technologies, exceptional specialized expertise and training in flow cytometry. Services include fluorescence-activated cell sorting (FACS), flow cytometric analysis, magnetic cell separation, automated cell counting and viability, consultation, data analysis and training. Access to instruments in the facility for fully trained users is 24 hours and 7 days a week.

MAJOR EQUIPMENT A 4 laser Millipore ImageStreamX MKII imaging flow cytometer providing a

multispectral image for every cell Six Flow Cytometric Cell Analyzers; 5 laser BD LSR Fortessa, 2 X 4 laser BD LSR IIs, 3 laser BD Canto II (violet), 3 laser BD RUO Canto II (yellow-green), and 4 laser Invitrogen Attune NxT

High Through-put Flow Cytometric Analysis; High through-put systems available on flow cytometric analyzers

Three Flow Cytometric Cell Sorters; 5 laser BD SORP Aria II, 4 Laser BD SORP Aria IIu and 4 Laser BD Aria IIu

Viability Analyzer; Beckman Coulter Vi-CELL Magnetic Cell Separator; Miltenyi AutoMACS Pro Cell Tissue Dissociator; Miltenyi gentleMACS Octo Tissue Dissociator Two Computer Work Stations; both Mac and PC

SERVICES Cellular Analysis: Assisted and unassisted flow cytometric and viability analysis using up to 5 separate lasers and 20 parameters for multiple assays including small particles.

Cell Sorting: Assisted and unassisted flow cytometric, and magnetic cell sorting services that include parity with analyzers so any project capable of analysis can be moved to cell sorting.

Data Analysis: Assisted and unassisted data analysis including a dedicated server for data storage, workstations for data analysis and a FlowJo software site license available to investigators.

Training: Training is available on all instrumentation including a Flow Analyzer training course as well as individual training on cell sorting and other instrumentation, Additionally, software or equipment updates upon request.

CORE LEADERSHIP

Christine Beeton, PhD

Academic Director

Associate Professor

Department of Molecular Physiology and Biophysics

713.798.5030

[email protected]

Joel M Sederstrom, MS

Core Director

713.798.3774

[email protected]


Oncogenic mTOR signalling recruits myeloid-derived suppressor cells to promote tumour initiation.

This work demonstrated that mTOR signaling in cancer cells dictates a mammary tumor’s ability to stimulate the accumulation of myeloid-derived suppressor cells through regulating G-CSF.

A, Flow cytometry quantification of tumor-infiltrating CD11b+Gr1+ cells. Animal numbers in each model are indicated. B-C, Flow cytometry quantification of CD11b+Gr1+ cells in peripheral blood. Animal numbers: B, tumor-free (TF): n = 4, MMTV-Wnt1: n = 5, MMTV-Wnt1 iFGFR: n = 7. C, TF: n = 4, P53N-A: n = 4, P53N-B: n = 2, P53N-C: n = 5. D, Flow cytometric analyses show that CD11b+ cells in blood of P53N-C tumor-bearing mice express Ly6C at a lower and more variable level compared to normal neutrophils. Upper: Representative data of Ly6GhighCD11b+ leukocytes in TF and P53N-C tumor-bearing mice. Lower: Ly6C histogram of CD11b+Ly6G+ cells in P53N-C tumor-bearing (dark) or TF (light) mice. E, In vitro T-cell proliferation, measured by CFSE assay, is inhibited by MDSCs. Histograms of CFSE levels in CD3- and IL-2 stimulated T-cells with or without co-culture of MDSCs at 1:3 ratio.

Welte T, Kim IS, Tian L, Gao X, Wang H, Li J, Holdman XB, Herschkowitz JI, Pond A, Xie G, Kurley S, Nguyen T, Liao L, Dobrolecki LE, Pang L, Mo Q, Edwards DP, Huang S, Xin L, Xu J, Li Y, Lewis MT, Wang T, Westbrook TF, Rosen JM, Zhang XH. Oncogenic mTOR signalling recruits myeloid-derived suppressor cells to promote tumour initiation. Nat Cell Biol. 2016, 18:632-644.


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DRUG DISCOVERY

CORE LEADERSHI P

The mission of the Drug Discovery Core (DCD) is to develop small-molecule probes, preclinical candidates, and drugs for researchers and clinicians. We are committed to helping investigators identify low-molecular-weight compounds that will further research and lead to new therapeutics. The DDC has several technologies to measure binding of user-supplied ligands to targets. In addition to providing consultation and expertise to test, screen, or select for small molecules that bind target macromolecules, the DDC also makes its instruments (ITC, NMR, Plate Reader, Lightcycler, etc.) available to trained, qualified users to operate independently. Compound collections can be screened in a high-throughput manner to identify novel hits. Affinity-based selection of millions of different molecules (generated with DNA-encoded technology) can be performed in a single tube to identify those that most tightly bind a tagged, purified protein. Follow-up validation and diversification of screening or selection hits can be performed using newly synthesized analogs based on available structure-activity relationship data. The DDC state-of-the-art hardware enables investigators to search the chemical diversity of commercially purchased and BCM developed compound libraries to identify small molecules that perturb biological activity with specificity and potency.

MAJOR EQUIPMENT Tecan Freedom Evo Robotics System Tecan Infinite M1000 Pro Plate Reader Tecan HydroSpeed Plate Washer Liconic STX 220 Incubator Thermo Scientific Multidrop Combi GE Healthcare Auto-ITC-200 Roche Lightcycler 480 Real-Time PCR HP D300 Digital Dispenser Cell Culture Hoods and Incubators Cell Counter SensiQ SPR NMR 600 and 800MHz

SERVICES High-throughput chemical screening with various assay types

Cell-based screens – Luciferase, MTS, etc. Protein-based screens – Alpha Screen, Thermofluor(protein melting), etc.

Consultation on assay development Training on instrumentation for use independently

LIBRARIES Chembridge Fragments 4000 Enzo Screen-Well Kinase Inhibitors 80 Enzo Screen-Well Phosphatase Inhibitors 33 Maybridge Ro3 Fragments 1895 MicroSource NatProd Collection 800 NCI Approved Oncology Set III 97 NCI Approved Oncology Set V 114 NCI Diversity Set III 1597 NCI Mechanistic Diversity Set 821 NCI Natural Product Set II 119 Otava Fragments 300 Sigma LOPAC 1280 Scynexis Malaria Box 400

Martin M. Matzuk, MD, PhD

Academic Director

Stuart A. Wallace Chair and Professor

Department of Pathology & Immunology

713.798.6451

[email protected]

Kimberly Kerlec, BS

Lab Manager

Department of Pathology & Immunology

[email protected]

713.798.8132

Kevin MacKenzie, PhD

Co-Director

Associate Professor

Department of Pathology &

Immunology

[email protected]

832-824-8155


Fig. 1. Affinity of PKG Iß for cGMP and Rp-cGMPS by surface plasmon resonance (SPR). (A-B), SPR experiments with hepta-his tagged PKG Iß CNB-B (219-351) interacting with cGMP and Rp-cGMPS. Overlaid experimental data for three replicates are shown in black and fitted model in orange. OneStep injections were performed with a SensiQ Pioneer FE, which titrates the analyte over 3-4 orders of magnitude in concentration. This creates gradients of 0-10 and 0-100 μM for cGMP and Rp-cGMPS, respectively. cGMP and Rp-cGMPS shows KD values of 108.2±0.7 nM and 8.8±0.3 μM, respectively. Modified from: Campbell, J. C., et al. (2016) Crystal structure of PKG Ibeta CNB-B:Rp-cGMPS complex reveals an apo-like, inactive conformation. FEBS Lett. 2016 Dec 3. doi: 10.1002/1873-3468.12505.

Figure 2: Identification of inhibitors of the BMP type 2 receptors. A) Coommassie stain of the purified recombinant BMP type 2 receptors, ACVR2A, ACVR2B, and BMPR2. B) Thermal shift assay results from the BMPR2 screen with the ENZO Kinase Inhibitor Library. C) Chemical structures for the BMPR2-binindg molecules, Apigenin and Quercetin. D) Use of an in vivo granulosa cell assay shows that Apigenin inhibits the GDF9:BMP15-induced expression of Has2. This data was provided by Dr. Diana Monsivais from Dr.Matzuk’s lab. Data in panel B was generated using the Roche Lightcycler 480.


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The mission of the Genomic & RNA Profiling (GARP) Core Facility is to provide the most cutting edge genomic technology to research investigators with an emphasis on personal service and quality. In order to provide whole genome profiling we offer next generation sequencing platforms (Illumina HiSeq2500 and MiSeq), microarray platforms, qPCR, targeted NanoString nCounter assays, and data analysis.

MAJOR EQUIPMENT Illumina HiSeq 2500 Sequencer Illumina MiSeq Benchtop Sequencer Illumina cBOT cluster generation instrument Nanostring nCounter Digital Quantification System Affymetrix Microfluidics 450 (2) and Scanner Agilent Scanner ABI ViiA7 Real Time PCR/qPCR instrument Agilent Bioanalyzer Covaris Ultrasonicator

SERVICES Next generation sequencing – RNA-seq (polyA, whole transcriptome, small RNA) – DNA-seq (whole genome and targeted capture) – ChIP-seq (protein-DNA interactions)

Nucleic Acid Shearing Microarray – Affymetrix expression and SNP – Agilent expression and aCGH – Custom array processing

Targeted NanoString nCounter assays (up to 800 multiplexed assays/sample)

Quantitative PCR Sample quality check Data analysis Consultation

GENOMIC & RNA PROFILING (GARP)

Lisa D. White, PhD

Academic Director

Associate Professor, Department of Molecular & Human Genetics

713.798.7607

[email protected]

Daniel C. Kraushaar, PhD

Technical Core Director

713.798.7787

[email protected]

CORE LEADERSHIP


MeCP2 binding to non-CG methylated DNA in the adult brain


A) Browser representation of mCG density in the adult mouse brain (black). Shown are ChIP-seq profiles for MeCP2, a methyl-CpG binding protein, from pluripotent ES cells (green), adult mouse brain (blue) and adult mouse hypothalamus (red).B) Correlation between MeCP2 binding profiles from three independent data sets. Correlation between data sets from adult brain (Upper). Correlation between data sets from ES cells and adult brain (Lower). r denotes Pearsons’s correlation.C) Enrichment of MeCP2 following ChIP-seq at repetitive DNA elements. Data were normalized and represented as the fold-change relative to Input DNA-seq controls.D) Verification of differences in MeCP2 binding by ChIP followed by RT-PCR.

Chen, L., Chen, K., Lavery, LA., Andrew Baker, S., Shaw, CA., Li, W., Zoghbi, HY. MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome. PNAS 2015 112(17):5509-5514.

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The Genetically Engineered Mouse (GEM) Core provides specialized expertise and equipment for essential mouse services. This includes microinjections of mouse ES cells, transgene DNA constructs, and CRISPR sgRNA/Cas9/donor DNA into mouse embryos to generate genetically engineered founder mice. The GEM Core also performs cryopreservation of mouse embryos and sperm for long-term storage of mouse lines; mouse line rederivation to remove pathogen contamination; re-establishment of mouse colonies; help with importation of mouse lines into BCM facilities; in vitro fertilization; and mouse colony expansion. Although the GEM Core can directly accept ES cells and CRISPR sgRNA/Cas9/donor DNA samples from investigators for microinjection, there is a separate mouse ES Cell Core that provides services for genetic manipulation of ES cells and preparation of CRISPR sgRNA/Cas9/donor DNA. The GEM and ES Cell Cores work together closely to provide comprehensive services.

MAJOR EQUIPMENT Nikon Eclipse Te300 Microscopes with Hoffman objective lenses Nikon Diaphot inverted microscopes Narishige micromanipulators SMZ 800 and 1000 dissecting microscopes Sutter P-97 horizontal pull Cryosafes SSBA LN2 storage units Osmometer

SERVICES Generation of transgenic mice by DNA microinjection (Traditional, BAC, and Lentiviral)

Mouse embryonic stem cell microinjection using C57BL/6J blastocysts from super ovulation

Mouse embryonic stem cell microinjection using Albino C57BL/6J (also known as B6 albino with mutated tyrosinase) blastocysts from natural mating

Mouse embryo cryopreservation Re-derivation of mouse lines Importing and exporting mouse lines Mouse line colony expansion Mouse sperm cryopreservation In vitro fertilization Generation of transgenic mice on existing mutant backgrounds

Microinjection of CRISPR gRNA/Cas9/ donor DNA

Storage of cryopreserved embryos and sperm

GENETICALLY ENGINEERED MOUSE (GEM)

Jianming Xu, PhD

Academic Director

Professor, Department of Molecular and Cellular Biology

713.798.6199

[email protected]

Lan Liao, M.S.

Core Director

Instructor, Department of Molecular and Cellular Biology

713.798.4684

[email protected]

CORE LEADERSHIP


Figure 1. Characterization of mice with inducible expression of miR-93 in podocytes. (a) Schematic design of the LB2-FLIP-GFP-miR-93Tg lentivirus used to generate floxed miR-93Tg mice. The construct was designed so that Cre induction could be used to mediate the inversion of pre-miR-93 into a sense orientation. (b) Immunofluorescence micrographs of kidney sections collected from control (sesame oil) or tamoxifen (Tam)-injected mTmG/.; Pod-CreERT2 reporter mice. (c) Flow cytometry analysis of kidney single cell suspension from control or Tam-injected mTmG/.; Pod-iCreERT2 mice. (d) Breeding scheme that was used to generate db/db:miR-93PodTg mice. (e) Top, representative genotyping PCR detecting transgenic miR-93, transgenic Pod-iCreERT2, and db alleles. Boxes highlight representative genotypes from mice carrying the three desired alleles. Bottom, an image of miR-93PodTg mice with or without Tam injections. (f) qPCR analysis for miR-93 expression levels in mouse podocytes isolated from control or Tam-injected miR-93PodTg mice. (g) Top, light micrographs of miR-93PodTg kidney sections stained with PAS from control or Tam-treated mice. Bottom, transmission electron micrographs of miR-93PodTg kidneys from control or Tam-treated mice. (h–k) Baseline phenotyping of control mice and Tam-injected miR-93PodTg mice for (h) body weight, (i) blood glucose, (j) ACR and (k) systolic blood pressure


Inducible expression of miR-93 in podocytes in transgenic mice

Badal SS, Wang Y, Long J, Corcoran DL, Chang BH, Truong LD, Kanwar YS, Overbeek PA, Danesh FR. miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy. Nat. Commun. 2016 7:12076, 6/28/2016

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The mission of the Gene Vector Core (GVC) is to assist investigators by providing gene transfer vectors which can be used for studying gene function by over-expression, ectopic expression, gene silencing, or gene editing. Recombinant viral vectors retain the native features of viruses which have been tested in nature for millions of years, and are among the most efficacious. The GVC has undertaken a variety of activities aiming at increasing productivity, improvement/development of quality control assays, and expanding the repertoire of viral vector-based research tools. The core offers a number of popular platforms and has extensive experience in the production of viral vectors including adeno-associated virus (AAV), first-generation (FGAd) and helper-dependent adenovirus (HDAd), lentivirus (LV) and retrovirus (RV).

SERVICES Packaging and purification of AAV (serotype 1, 2, 5, 6, 7, 8 and 9, and hybrid DJ and DJ8), in half-scale, regular-scale or in high quality large scale.

Rescue, and/or amplification/purification of FGAd (serotype 5) and HDAd (serotype 2, 5 and 5/35, and custom targeted vectors).

Packaging and concentration/purification of VSVG-pseudotyped LV with 2nd or 4th generation packaging systems.

Packaging RV (ecotropic and amphotropic). Subcloning into viral transfer vectors and preparation of plasmids for viral vector production.

Titration for infectivity. Customer provides transfer vectors for transfection. Packaging plasmids or helper viruses are provided by the Core.

Off-the-shelf packaged vectors are available. Common viral transfer plasmids vectors developed by the Core are available.

GENE VECTOR

CORE LEADERSHIP

Kazu Oka, PhD

Core Director

Associate Professor, Molecular and Cellular Biology

713.798.4478

[email protected]


Singh VP, Mathison M, Patel V, Sanagasetti D, Gibson BW, Yang J, Rosengart T. MiR-590 promotes transdifferentiation of porcine and human fibroblasts toward a cardiomyocyte-like fate by fiercely repressing specificity protein 1. J Am Heart Assoc 2016 (Epub ahead of print)

Differentiated human cells are relatively resistant to reprograming, perhaps due to epigenetic stabilization. Singh et al found that Gata4 (G), Mef2c (M) and Tbx5 (T) induces cardiomyocyte marker, cardiac troponin T (cTnT) in rat fibroblasts, while porcine and human fibroblasts require addition of Hand2 (H) + Myocardin (My), and/or miR-590. When co-cultured with mouse cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions.


Reprograming of Porcine Cardiac Fibroblasts

In the above figure porcine cardiac fibroblasts were transduced with lentivirus co-expressing GFP and co-cultured with mouse cardiomyocytes. A) Immunofluorescence staining. B) Contraction and Ca2+ transient. Contractions of myocytes from random fields were videotaped and digitized on a computer. Calcium transients were measured after loaded with Fura-2 for 30 min. C) Representative Immunofluorescence. DAPI (blue), GFP (green, lenti infection marker) and -actinin (red, cardiomyocyte marker).

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The Human Tissue Acquistion and Pathology (HTAP) Core provides services for processing of tissues for various histological and immunohistochemical (IHC) assays for research projects of investigators working with either human specimens or animal models. Services are performed by expert technical staff with assistance of pathologists who provide consultation and review of slides and images. Histology, tissue microarray development, immunohistochemistry, laser capture microdissection and image analysis are available on a fee-for-service basis. HTAP is also involved in tissue acquisition and tissue banking. The tissue bank functions to provide human tissue to BCM researchers that have IRB protocols in place. Tissue requests can be made by contacting the Core Director and are then reviewed by committee.

MAJOR EQUIPMENT Shandon Excelsior ES Tissue Processor Shandon HistoCentre Embedding System Sakura TissueTek SCA Coverslipper Shandon Varistain Gemini Slide Stainer Microm HM 315 Microtome Microm 505 E Cryostat Nuance FX multispectral imaging system Arcturus XT Laser Capture Microdissection instrument Vectra imaging system Nikon slide scanning and imaging system

SERVICES Histology - Tissue processing, embedding, cutting, and staining of human and animal tissues

Immunohistochemistry (IHC) and TUNEL Assays - IHC for proliferation and apoptosis are performed using optimized methods and antibodies provided by the Core. Investigator supplied antibodies are used for other IHC assays which are optimized for performance.

Laser capture microdissection (LCM)-Isolation of specific cell types or group of cells from frozen of paraffin tissue sections to be used for RNA, DNA or protein analyses.

Digital imaging - State-of-the-art imaging of tissue sections or TMAs using the Nikon slide scanner or Vectra imaging system with Nuance FX multispectral camera. Image analysis using inForm software or Nikon Elements technology for pattern recognition analysis and quantitative scoring.

Tissue microarray (TMA) - TMAs are developed using the Cores archival formalin fixed paraffin embedded sections or tissues provided by researchers.

Consultation with pathologists. Experienced pathologists will assist with review of stained slides.

HUMAN TISSUE ACQUISITION AND PATHOLOGY (HTAP)

Michael Ittmann, MD, PhD

Academic Director

Professor, Department of Pathology & Immunology

713.798.6196

[email protected]

Patricia Castro, PhD

Core Director

Instructor, Department of Pathology & Immunology

713.798.6795

[email protected]

CORE LEADERSHIP


The above figure is taken from Dr. Heaney’s PNAS paper cited below (PMID: 25918379). The figure shows that IL-33 (green) expression pattern in the normal intestinal mucosa (Top) and a polyp of ApcMin/+ mice (Bottom). IL-33 immunoflourescence was used with CTNNB1, a marker from mucosal epithelium; (Panel A) and with ACTA2, a marker for stromal cells (Panel B) to show localization of IL-33 positive cells with the context of the tissue. IL-33 is localized to the stromal cells of the normal intestinal but is expressed in the transformed epithelial cells of polyps. The IL-33 promotes intestinal tumorigenesis in ApcMin/+ mice and activates two stromal cell types, subepithelial myofibroblasts and mast cells, known to mediate intestinal dysplasia and create a microenvironment favorable to tumorigenesis. The mouse tissues used for these studies were processed and sectioned by the HTAP histology lab.

Maywald RL, Doerner SK, Pastorelli L, DeSalvo C, Benton SM, Dawson EP, Lanza DG, Berger NA, Markowitz SD, Lenz HJ, Nadeau JH, Pizarro T, Heaney JD. IL-33 activates tumor stroma to promote intestinal polyposis. Proc Natl Acad Sci USA 2015; 112(19):


IL-33 localizes to transformed epithelial cells in polyps and SEMFs in normal mucosa.

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The Human Stem Cell Core (HSCC) provides a wide range of products and services related to human pluripotent stem cell research, as well as hands-on training classes for basic and advanced stem cell culture techniques. We offer cost-effective solutions to generate and characterize novel induced pluripotent stem (iPS) cells for in vitro disease modeling, employing “zero-footprint” technologies such as Sendai virus-based and episomal vector-based reprogramming. We also offer customized research support for experimental design and validation assays.

MAJOR EQUIPMENT EVOS XL and FL inverted microscope systems Lonza 4D-Nucleofector transfection system Beckman Coulter Allegra X-14R centrifuge ABI StepOnePlus Real-Time PCR system MVE TEC 3000 LN2 cryostorage system

SERVICES Hands-on training classes and workshops Human pluripotent stem cell (ESC and iPSC) culture services

Reprogramming of human somatic cells to iPS cell lines

Stem cell line characterization Mycoplasma testing Consultation on experimental design

HUMAN STEM CELL

CORE LEADERSHIP

Margaret Goodell, PhD

Academic Director

Professor

Pediatrics-Hematology

713.798.1230

[email protected]


Jean J. Kim, PhD

Core Director

Assistant Professor

Department of Molecular & Cellular Biology

713.798.7471

[email protected]

A) Immunofluorescence staining for OCT4 and TRA-1-81 in the original 003 fibroblasts (upper panels) and iPSC clone 5 (lower panels). B) qRT-PCR for OCT4, SOX2, NANOG, and REX1 in 003 fibroblasts and iPSC clones 5 and 8. C) Analysis of iPSCs by flow cytometry show >95% of iPSCs are double-positive for OCT4 and SSEA4. D) Analysis by G-banding shows a normal male karyotype..

Unpublished results. Immunofluorescence and qRT-PCR data courtesy of Jordan Kho in Dr. Brendan Lee’s lab.

Basic characterization of M22 human induced pluripotent stem cell (iPSC) lines generated from skin fibroblasts by the HSCC.


The Integrated Microscopy Shared Resource is a state-of-the-art imaging, training and assay development resource to support live and fixed cell confocal, deconvolution and super-resolution microscopy, transmission electron microscopy, and fully-automated high throughput microscopy. A full suite of image analysis, statistics and reporting software is available for data mining and management.

MAJOR EQUIPMENT Nikon A1-Rs spectral confocal microscope with live cell incubator

GE Healthcare DeltaVision deconvolution microscope with large sCMOS camera and live cell incubator

Nikon Ci-L upright brightfield microscope with color camera Vala Sciences IC-200 high throughput microscope for live and fixed samples in multi-well plates

Beckman BioMek NX microfluidics robots Hitachi H-7500 and JEOL1230 digital transmission electron microscopes

GE Healthcare OMX Blaze super-resolution instrument (SIM, dSTORM) with ultrafast camera for live imaging and TIRF capabilities

Biotek Cytation 5 microscope-in-a-box (fluorescence, color, slide scanning, plastic plates, live imaging, hypoxia), plus plate reader (fluorescence, absorbance, luminescence)

SERVICES One-on-one training for all instruments Assay development and project consultations Fully automated and assisted high throughput microscopy for 96/384 well plates

Image Analysis, custom or pre-set (i.e., cell count, subcellular localization, spot counting, translocation, cell cycle, toxicity, live/dead, apoptosis)

Full service processing, sectioning and scoping for transmission electron microscopy

Training in immunofluorescence and RNA FISH protocols

INTEGRATED MICROSCOPY CORE (IMC)

Michael Mancini, PhD

Academic Director

Professor, Molecular and Cellular Biology

713.798.8952

[email protected]

CORE LEADERSHIP

Fabio Stossi, PhD

Core Director

Assistant Professor, Molecular and Cellular Biology

713.798.6940

[email protected]


Figure Legend: 3D reconstruction (Z-axis projection) of a natural killer cell imaged on the GE Healthcare OMX 3D-Structured Illumination Microscope (SIM). The actin cytoskeleton is stained with phalloidin-AF568. This image highlights the abundance of actin branches (filopodia) and uses false colors to represent the height of the structures.

Natural Killer Cell Actin Cytoskeleton Imaged by 3D Structured Illumination Microscopy (SIM)

Unpublished data courtesy of Dr. Alexandre Carisey and Dr. Jordan Orange, Center for Human Immunobiology, Baylor College of Medicine, Texas Children’s Hospital.

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Mass Spectrometry Proteomics Core offers services for quantitative proteome-wide profiling of cells and tissues, affinity isolated protein complexes, post-translational modification (PTM) analysis, and routine identification of purified proteins. We specialize in providing comprehensive project-based support that includes project design, optimization of biochemical procedures for sample preparation, state-of-the-art mass spectrometry technology, and custom data analysis to address specific challenges of different proteomics approaches.

MAJOR EQUIPMENT Thermo Scientific Orbitrap Elite, Q-Exactive Plus, Orbitrap Fusion Tribrid, and Orbitrap Lumos ETD Tribrid Mass Spectrometers with EASY-nLC1000/1200 or Dionex Ultimate 3000 RSLCnano UHPLC Systems

SERVICES 365 Proteome Profiling service combines efficient non-detergent sample preparation procedure with dual reverse phase fractionation procedure and optimized mass spectrometry acquisition methods to allow identification and label-free quantification of up to 6,000 proteins from as few as 100,000 cells or 20 micrograms of protein lysate.

IP/MS (antibody-based purification followed by MS identification) packages offer characterization of protein complexes and their extended interaction networks. The core’s unique emphasis is in purification of endogenous complexes. Custom data analysis against BCM’s own complexome database and filtering of non-specific precipitants is included in this package service.

Post-translational modification (PTM) analysis includes identification and quantification of phosphorylation, ubiquitination or acetylation sites on purified proteins.

The core offers routine MS sequencing of purified protein samples for single-protein identification or verification.

CORE LEADERSHIP

Anna Malovannaya, PhD

Academic Director

Assistant Professor, Department of Biochemistry and Molecular Biology

713.798.8699

[email protected]

Sung Yun Jung, PhD

Core Director


713.798.7231

[email protected]

Hon-chiu Eastwood Leung, PhD

Co-Director

Assistant Professor, Departments of Pediatrics and Molecular and Cellular Biology

713.798.6360

[email protected]

MASS SPECTROMETRY PROTEOMICS



IP/MS identifies the spliceosome as a therapeutic vulnerability in MYC-driven cancer.

Dr. Thomas F. Westbrook’s laboratory used the MS Proteomics core to find a new regulatory mechanism of c-MYC protein, one of the most common drivers of human cancer. Delineating how oncogenes such as MYC confer cellular stresses and how these stress phenotypes are tolerated in cancer cells is an area of emerging interest. In affinity purification of MYC complex, we identified BUD31 as one of the interacting proteins. Furthermore, a reciprocal purification and sequencing of Flag-tagged BUD31 complex confirmed that spliceosomal components were associated with BUD31 and c-MYC. In follow-up studies, Dr. Westbrook’s laboratory showed that BUD31 is a MYC-synthetic lethal gene and demonstrated catalytic activity. Using these orthogonal approaches, the spliceosome was shown to be required to tolerate oncogenic MYC. Importantly, Dr. Westbrook’s group showed that genetic and pharmacologic inhibition of the spliceosome severely impairs aggressive breast cancers driven by the MYC oncogene.

Hsu TY, Simon LM, Neill NJ, Marcotte R, Sayad A, Bland CS, Echeverria GV, Sun T, Kurley SJ, Tyagi S, Karlin KL, Dominguez-Vidaña R, Hartman JD, Renwick A, Scorsone K, Bernardi RJ, Skinner SO, Jain A, Orellana M, Lagisetti C, Golding I, Jung SY, Neilson JR, Zhang XH, Cooper TA, Webb TR, Neel BG, Shaw CA, WestbrookTF. The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature. 2015;525(7569):384-388

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METABOLOMICSThe Metabolomics Core provides targeted and unbiased metabolic profiling for discovery and validation of biomarkers of various diseases with state-of-the-art high throughput mass spectrometry as the main platform. Metabolites can be measured in tissue samples, cell lines, fecal and biofluids including urine. The entire process starting from sample preparation to mass spectrometry is monitored using spiked isotopic standards that have been characterized for their chromatographic behavior as well as fragmentation profile. Biostatisticians are available for further analysis of the resulting output data.

TECHNOLOGIES

Targeted metabolite steady-state profiling: The Core has the capability of identification, quantification and characterization of over 500 metabolites using the targeted multiple reaction monitoring approach (MRM) developed for different chemical classes of compounds. Data can be reported either in absolute concentrations or as intensity ratios to internal standards.

Metabolic Flux: Isotope flux and metabolite profiling to help formulate and test hypotheses about the metabolic consequences of various changes, in order to guide further integrative systems biology analyses of underlying mechanisms in disease. The Core has the capability of characterization of [13C] Glutamine and [13C]Glucose flux using LC-QQQ and LC-QTOF Mass Spectrometry.

Lipidomics: Using an ABSCIEX 5600 triple TOF MS, identification of lipids is accomplished by data-dependent production (MS/MS) information of human plasma, tissues, and urine in both positive and negative ionization modes. MS/MS acquisition or MS/MS ALL acquisition provides information on the nature of the lipid head group and/or neutral loss of the head group from the molecular ion adducts. Information on fatty acid composition of the lipids is obtained in the negative mode.

Drug metabolism and pharmacokinetics: We provide rapid and efficient approaches to investigate metabolism, metabolic stability, pharmacokinetics, and chemical structure identification of small molecular weight compounds using liquid chromatography-mass spectrometry (LC-MS and MS/MS).

CORE LEADERSHIP

Nagireddy Putluri, PhD

Core Director

Associate Professor, Department of Molecular & Cellular Biology

713.798.3139

Arun Sreekumar, PhD

Academic Director

Professor, Department of Molecular & Cellular Biology

713.798.3305


E QUIPMENT & SERVICES

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer


MAJOR EQUIPMENT Agilent 6490 triple quadruple (QQQ) Mass Spectrometers Agilent 6550 QTOF UHD Accurate-Mass Spectrometer AB SCIEX 5600 Triple TOF Mass Spectrometer 1290 and 1260 Series HPLC Systems

SERVICES Mass determination Metabolic identification by tandem MS Metabolic Profiling Absolute Quantification of Metabolites Metabolic Flux Measurements Pathway mapping using OCM, GSA or NETGSA Developing classification models Integration with other OMICS datasets

CLASSES OF METABOLITES MEASURED BY MRM ANALYSIS

1. Amino acids and related metabolites 2. Amino Sugars 3. Lipids 4. Prostaglandins 5. Carnitines 6. Polyamines 7. TCA metabolites 8. CoA’s 9. Carnitines 10. Sugars 11. Nucleotides/Nucleic acids 12. Vitamins 13. Steroids 14. Neurotransmitters, 15. Poly cyclic aromatic hydrocarbons 16. Miscellaneous

Transmitochondrial cybrids, cell lines, PDX models and clinical data combined with multiple OMICs approaches to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple negative breast cancer (TNBC). In this study the Metabolomics core used a combination of steady state metabolomics to define the increased mitochondrial fatty acid -oxidation (FAO) metabolites in TNBC [1]. The data showed that metastatic TNBC is dependent on FAO energy metabolism and metastatic TNBC cell lines have increased carnitine levels compared to benign breast cell lines. FAO also activated the Src oncopathway, one of the commonly upregulated pathways in TNBC by its autophosphorylation at Y419. FAO inhibition decreased in vitro and in vivo TNBC growth and metastatic progression. These results provided insights to the significance of mitochondrial energy reprogramming to tumor properties, and the regulation of a major cancer pathway in TNBC.

A) Hypothetical model showing the regulation of Src oncopathway by energy reprogramming. Metastatic triple negative breast cancer (TNBC) show high energy dependency to mitochondrial fatty acid -oxidation (FAO). FAO activates c-Src oncoprotein via autophosphorylation of Src at Y419. Src phosphorylation correlated with mRNA expression of the FAO rate-limiting CPT1 gene. Inhibition of FAO decreased TNBC progression and metastasis. B) Mass spectrometric analysis of FAO metabolites in benign and metastatic TNBC cells showing increased carnitine levels in TNBC cells (MDA231 and SUM159) compared to benign cells (A1N4).

Park, J.H., et al., other authors Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer. Cell Rep, 2016. 14(9): p. 2154-65.

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MHC Tetramer technique has become a “gold standard” for the quantification of T cell immune responses. Joining multiple copies of the MHC/antigen complex into a single probe resolves the difficulties presented by the low affinity of the class I MHC molecule for the CD8 receptor. By offering exquisite antigen specificity and sensitivity, this unique technique is suitable for basic and clinical studies in a number of applications, including cancer prevention, cancer therapy, cell and gene therapy, immunotherapy, and non-cancer related immunology research. This technique is U.S. patent protected. The mission of the core is to provide BCM investigators with customized MHC/peptide tetramers for identification of antigen specific T lymphocytes by flow cytometry.

SERVICES MHC Class I Tetramers We offer more than fifty human, mouse, macaque, and chimpanzee alleles for customized production of class I MHC reagents with desired epitopic peptides. Researches will also have two fluorescent labels choices: R-phycoerythrin (PE) or allophycocyanin (APC).

Biotinylated Monomers For customers who intend to try to label tetramers with small molecule fluorophores or require longer storage life

Unbiotinylated Monomers Can be used in various applications such as ELISA or ELISPOT;

using unbiotinylated monomers coat the plate to present the peptide Special reagents – CD8 binding-deficient MHC Class I tetramers – MHC Monomers for generating TCR-like antibodies

MHC TETRAMER

CORE LEADERSHIP

Lily Wang, MS

Core Director

713.798.3918

[email protected]


The example study focused on the energy sensor AMPK (AMP-activated protein kinase) dependent T cell metabolism and T cell-mediated adaptive immunity. The influences of nutrient availability on the T cell metabolism and function is always poorly understood. This study found that activated T cells possessed a glucose sensitive metabolic checkpoint controlled by the AMPK that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. They also demonstrated that AMPKa1 is essential for Th1 and Th17 cell development and primary T cell responses to viral and bacterial infections. Our reagents H-2KbOVA257-264 tetramer and H-2Db-NP tetramer were utilized in this study.

In figure A, control wild type (WT-T) and AMPKa1-deficient (KO-T) mice were infected with pulmonary influenza A virus. Then splenocytes were stained with #2 tetramer at day 9 after post-infection. In figure B, WT-T and KO-T mice were infected with L. Monocytogenes. Splenocytes were stained with #1 tetramer at day 7 after post-infection. The frequency and total number of tetramer+/CD8+ T cells are indicated. Both figures showed a decrease of the frequency of tetramer+ CD8+ T cells in the KT-O mice, which demonstrated AMPKa1 is required for primary T cell response.

Blagih J, Coulombe F, Vincent EE, Dupuy F, Galicia-Vazquez G, Yurchenko E, Raissi TC, Van der Windt GJW, Viollet B, Pearce EL, Pelletier J, Piccirillo CA, Krawczyk CM, Divangahi M, and Jones RG. The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo. Immunity. 2015; 42(1):41-54. PMID: 25607458


The mission of the Mouse Embryonic Stem Cell Core is to provide BCM investigators with standardized, high quality services for precise engineering of the mouse genome. Procedures for modifying the genome of mouse embryonic stem (ES) cells and the subsequent construction of mice from manipulated ES cells are intensive and technically demanding. It is a goal of the Core to make such experiments accessible to a wide range of BCM investigators. The Core focuses on the manipulation of embryonic stem cells for further studies in vitro with primary mouse ES cell lines and in vivo with mice generated with targeted ES cells. In addition, the Core provides centralized, highly specialized expertise in the design and rapid production of reagents for in vivo CRISPR/Cas9 genome engineering in mice (and other model organisms such as rats) and for in vitro CRISPR/Cas9 mutagenesis in mouse ES cells. The Mouse ES Cell Core works closely with the Genetically Engineered Mouse (GEM) Core by providing targeted ES cells for blastocyst microinjections and by providing CRISPR/Cas9 reagents for microinjection of pronuclear stage embryos.

SERVICES Advice with construct design for gene targeting in ES cells Electroporation of mouse ES cells (129 or B6-derived) with targeted vectors provided by investigators

Isolation of ES cell colonies for genotyping Expansion of mouse ES cell clones from single wells of 96 well plates

Cryopreservation of mouse ES cells Transgene knock-ins at the Rosa26 and Hprt loci Advice with KOMP/EUCOMM vectors and ES cells Generation of new mouse ES cell lines from blastocysts Murine virus testing of ES cells In vivo CRISPR/Cas9 mutagenesis project design and reagent production for both nonhomologous end joining and homology directed repair in mice (and other model organisms such as rats)

CRISPR/Cas9 mutagenesis in mouse ES cells CRISPR/Cas9 on target and off target genotyping

Jason Heaney, PhD

Academic Director

Assistant Professor, Molecular and Human Genetics

713.798.1778


Isabel Lorenzo

Core Director

Instructor, Department of Molecular and Human Genetics

713.798.1981


Targeting the spermatogenesis-associated protein 7 gene

The mouse Spata7 gene was deleted using ES cell gene targeting to elucidate the mechanisms by which Spata7 contributes to leber congenital amaurosis and juvenile retinitis pigmentosa. A) A knockout mouse model of Spata7 deficiency was generated by replacing exons 1 through 10 of Spata7 in 129 ES cells with a Neo cassette. ES cells were then used to generate chimeric mice and a Spata7 knockout line. B) Spata7 deficiency causes progressive photoreceptor degeneration in the retina. By 6 months of age a marked reduction in the thickness of the outer nuclear layer (ONL) of the retina is observed in Spata7 mutant mice (-/-) when compared to Spata7 wild-type controls (+/+).

Eblimit A, Nguyen TM, Chen Y, Esteve-Rudd J, Zhong H, Letteboer S, Van Reeuwijk J, Simons DL, Ding Q, Wu KM, Li Y, Van Beersum S, Moayedi Y, Xu H, Pickard P, Wang K, Gan L, Wu SM, Williams DS, Mardon G, Roepman R, Chen R. Spata7 is a retinal ciliopathy gene critical for correct RPGRIP1 localization and protein trafficking in the retina. Hum Molec Genet. 2015, 24(6):1584-1601.

MOUSE EMBRYONIC STEM CELL CORE

A

B

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MOUSE METABOLISM CORE (MMC) The MMC supports investigators in state-of-the-art analysis of metabolic pathways and related physiological and biochemical parameters in mice and rats, as well as in isolated/cultured cells in vitro. Gene targeting and transgenesis have produced a large number of genetic mouse models for metabolism research. The MMC employs sophisticated assays and procedures to characterize both genetic and nongenetic rodents in a cost-effective manner. Dr. Saha personally performs or supervises all procedures in the core. Directors provide consultation on selecting the appropriate tests and procedures, and with the interpretation of data.

MAJOR EQUIPMENT XF 24 Analyzer (Seahorse instrument) Comprehensive Laboratory Animal Monitoring System (CLAMS) EchoMRI-100™ Versamax system Syringe pump for perfusion Rectal probe for body temperature measurement Modular treadmill (single lane 4 modular treadmill with shock)

SERVICES Hyperinsulinemic-euglycemic clamp in conscious mice: This is a gold standard for metabolic & diabetes research. Several key parameters such as HGP, BGP, GDR, tissue specific glucose uptake etc., are quantified in conscious mice. In addition, inclusion of appropriate isotopes will enable the direct quantification of the role of specific organs and tissues in glucose-insulin homeostasis in vivo.

Cellular oxygen consumption & glycolysis monitoring: Use of the Seahorse instrument enables the MMC to measure OCR & ECAR from cultured cells within minutes.

Metabolic monitoring: Simultaneous monitoring of food intake, energy expenditure and real-time body temperature for small animals (mouse) after 72 hours of acclimation using the Comprehensive Laboratory Animal Monitoring System (CLAMS).

Body composition analysis by MRI Metabolic Modular Treadmill for mouse exercise and energy expenditure.

Home cage activity monitoring. Glucose metabolism – Glucose tolerance test – Insulin tolerance test – Pyruvate tolerance test (gluconeogenesis)

Pradip Saha, PhD

Core Director

Assistant Professor, Molecular and Cellular Biology/Medicine

713.798.3704 [email protected]

CORE LEADERSHIP

Benny Hung-Junn Chang, PhD

Academic Director

Assistant Professor, Department of Molecular and Cellular Biology

713.798.6686

[email protected]



Adipose tissue macrophages underlie insulin resistance in high fat-fed diabetic mice.

A) Scheme for determining impact of ablation of PI-producing macrophages on glucose metabolism. B) Basal glucose production (GP), GP after clamp, glucose infusion rate (GIR), and glucose disposal rate (GDR) and C) Glucose uptake into gonadal adipose tissue (GWAT), inguinal white adipose tissue (IWAT), and soleus muscle. D-E) Lipoprotein profiles from non-transplanted (black) and chimeric mice repopulated with Ctr (green) and FH (red) human hepatocytes, F-H) Detection of lipoprotein a (LPA) and cholesteryl ester transfer protein (CETP), which don’t exist in mice.

Buras ED, Yang L, Saha P, Kim J, Mehta P, Yang Y, Hilsenbeck S, Kojima H, Chen W, C. Smith W, Chan L. Proinsulin-producing, hyperglycemia-induced adipose tissue macrophages underlie insulin resistance in high fat-fed diabetic mice. FASEB J.2015 May 7.15-271452

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The mission of the Mouse Phenotyping Core (MPC) is to provide standardized, high quality phenotyping services for mouse experimental models developed by research investigators at BCM. The Core provides state-of-the-art instrumentation to analyze a wide range of physiological parameters in mice and other small rodent models. Services include comprehensive imaging and non-imaging analyses. Imaging services can be performed on animals from the embryonic stage through adulthood thus minimizing the number of animals required for a project and animals can be housed in the Taub animal facility for longitudinal studies. The Core also provides assistance with protocol and experimental design, data analysis and training so that researchers have the opportunity for unassisted operation of the many pieces of Core equipment. The Mouse Phenotyping Core complements the Genetically Engineered Mouse (GEMs) Core.

MAJOR EQUIPMENT Vevo 2100 Ultrasound (Visulasonics) 7.0T Pharmascan MRI (Bruker) eXplore CT 120 (TriFoil Imaging) Ms-FX Pro Optical Imager and X-Ray (Bruker) Unrestrained Whole Body Plethysmography (Buxco) Oxymax FAST Indirect Calorimetry System (Columbus Instruments) 16 station Comprehensive Lab Animal Monitoring System [CLAMS] w/body temp implants (Columbus Instruments)

Piximus Bone Densitometer (Lunar) Non-Invasive Blood Pressure (IITC Life Sciences) Blood Pressure and ECG Telemetry (DSI) 6-lane treadmill (Columbus Instruments) and running wheels (minimitter)

16-Metabolic cages (VWR) ECG-Mouse Monitor (Indus Instruments) Pulse oximetry (Indus Instruments) Grip strength meter (Columbus Instruments)

SERVICES/PROCEDURES Transverse Aortic Constriction (TAC) Vessel Doppler (Indus Instruments) Telemetry Device Implantation Osmotic Pump Implantation Cecal Ligation Myocardial Infarction (In development)

MOUSE PHENOTYPING

CORE LEADERSHIP

Corey Reynolds, PhD

Core Director

Associate Professor, Department of Molecular Physiology & Biophysics

713.798.5040

[email protected]


Effects of low dose rapamycin on cardiac function. 6–8-week-old C57/Bl6J mice were treated for 4 weeks with rapamycin (10 g/kg), and cardiac function was measured using echocardiography once per week. A, average ejection fraction before rapamycin treatment. B, ejection fraction over time as a percentage of the initial measurement. C, average fractional shortening before rapamycin treatment. D, fractional shortening over time as a percentage of the initial measurement. E, average left ventricular internal diameter at diastole before rapamycin treatment. F, left ventricular internal diameter at diastole over time as a percentage of the initial measurement. G, average left ventricular internal diameter at systole before rapamycin treatment. H, left ventricular internal diameter at systole over time as a percentage of the initial measurement. Data are shown as mean ± S.E. (error bars).

Lee, C.S., Georgiou, D., Dagnino-Acosta, A., Xu,J., Ismailov, I., Knoblauch, M., Monroe, T., Ji, R., Hanna ,A., Joshi, A. Long, C., Oakes, J., Tran,T., Corona,B., Lorca,S., Ingalls, C., Narkar, V., Lanner,J.,Bayle,J., Durham, W. and Hamilton, S. Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function. J Biol Chem. 2014 Sep 12;289(37):25556-70. doi: 10.1074/jbc.M114.586289


LIGANDS FOR FKBP12 INCREASE CA2+ INFLUX AND PROTEIN SYNTHESIS TO IMPROVE SKELETAL MUSCLE FUNCTION

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The mission of the Optical Imaging and Vital Microscopy (OIVM) Core is to provide state-of-the-art instrumentation and cutting edge imaging and analysis tools for the research applications of a broad range of BCM investigators. This core is dedicated to vital and intravital imaging of processes within cells, intact tissue explants, developing embryos and functioning organs within the live animal. Our users are focused on a variety of applications such as understanding cell migration, optimizing angiogenic therapies, how blood flow influences development and cancer, immune cell recruitment, stem cell-niche interactions and cancer metastasis.

MAJOR EQUIPMENT Carl Zeiss LSM 780 – Confocal point scanning microscope Leica TCS SP8 – Confocal and two-photon microscope Carl Zeiss LSM 510 META – Confocal point scanning microscope Carl Zeiss LSM 5 LIVE – Confocal line scanning microscope Carl Zeiss LSM 7 MP – Two-photon point scanning microscope Carl Zeiss Lightsheet Z.1 – Light-sheet fluorescence microscope Bioptigen Optical Coherence Tomography (OCT) Microscope Carl Zeiss Axio Zoom.V16 Stereomicroscope Bruker Skyscan 1272 X-ray MicroCT Optical Projection Tomography (OPT) Microscope Dell Precision T7600/T7610 Workstations Dell PowerVault data storage array Allentown Nexgen 70 cage mouse rack Allentown Phantom Animal Transfer Station Thermo Scientific, Series Class II Tissue Culture Hood

SERVICES Multispectral 4D fluorescence confocal for imaging multiple vital dyes or autofluorescence reduction

Automated multiple field-of-view 3D confocal to image multiple specimens simultaneously

Live imaging of tissue/organ development in embryos and live animals with environmental control of the stage and anesthesia support of live animals

Live imaging of blood and fluid flow such as fluorescent beads, fluorescently labeled erythroblasts, etc.

Automated high resolution mosaic imaging using tile scan based on centered grid, rectangular grid, and convex hull grid methods

Light-sheet fluorescence microscopy for 3D imaging of thick tissues cleared with Scale, CLARITY, etc.

Live 3D imaging of tissues with minimal light scattering using light-sheet microscope

Imaging of highly scattering 3D cell cultures via multiview imaging and dual side illumination using the light-sheet microscope

Two-photon and second harmonic imaging for intravital imaging

Live imaging of mouse cornea with the OCT microscope

Imaging and 3D rendering of embryos, organs, bioengineered gels, etc. using the X-ray microCT scanner

Imaging and 3D rendering of optically cleared (BABB) embryos, mammary glands, etc. using the OPT microscope

Quantitative analysis of cellular dynamics and cell tracking

Custom design of image analysis

Mary E. Dickinson, PhD

Academic Director

Professor, Department of Molecular Physiology and Biophysics

713.798.2104

[email protected]

CORE LEADERSHIP

Tegy J. Vadakkan, PhD

Core Director

Instructor, Department of Molecular Physiology and Biophysics

713.798.6486

[email protected]

Irina V. Larina, PhD

Co-Director

Assistant Professor, Department of Molecular Physiology and Biophysics

713.798.3974

[email protected]

OPTICAL IMAGING AND VITAL MICROSCOPY (OIVM)



This imaging method is ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality (KOMP Grant).

Dickinson ME, Flenniken AM, Ji X, Teboul L, Wong MD, White JK, Meehan TF, Weninger WJ, Westerberg H, Adissu H, Baker CN, Bower L, Brown JM, Caddle LB, Chiani F, Clary D, Cleak J, Daly MJ, Denegre JM, Doe B, Dolan ME, Edie SM, Fuchs H, Gailus-Durner V, Galli A, Gambadoro A, Gallegos J, Guo S, Horner NR, Hsu CW, Johnson SJ, Kalaga S, Keith LC, Lanoue L, Lawson TN, Lek M, Mark M, Marschall S, Mason J, McElwee ML, Newbigging S, Nutter LM, Peterson KA, Ramirez-Solis R, Rowland DJ, Ryder E, Samocha KE, Seavitt JR, Selloum M, Szoke-Kovacs Z, Tamura M, Trainor AG, Tudose I, Wakana S, Warren J, Wendling O, West DB, Wong L, Yoshiki A; International Mouse Phenotyping Consortium.; Jackson Laboratory.; Infrastructure Nationale PHENOMIN, Institut Clinique de la Souris (ICS).; Charles River Laboratories.; MRC Harwell.; Toronto Centre for Phenogenomics.; Wellcome Trust Sanger Institute.; RIKEN BioResource Center., MacArthur DG, Tocchini-Valentini GP, Gao X, Flicek P, Bradley A, Skarnes WC, Justice MJ, Parkinson HE, Moore M, Wells S, Braun RE, Svenson KL, de Angelis MH, Herault Y, Mohun T, Mallon AM, Henkelman RM, Brown SD, Adams DJ, Lloyd KC, McKerlie C, Beaudet AL, Bucan M, Murray SA, High-throughput discovery of novel developmental phenotypes, Nature, 2016, 537(7621):508-514.

Hsu CW, Wong L, Rasmussen TL, Kalaga S, McElwee ML, Keith LC, Bohat R, Seavitt JR, Beaudet AL, Dickinson ME, Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages, Developmental Biology, 2016, 419(2):229-236.

3D rendering of early postnatal to early post-implantation mouse samples imaged by iodine contrast microCT. (P3-E9.5)

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The Patient-Derived Xenograft and Advanced In Vivo Models Core is divided into two independent but closely interacting functional units, Mouse PDX Development using immunocompromised mice as the host species, and Advanced In Vivo Models using patient material and cell lines grown on the chorioallantoic membrane of the chicken egg (CAM-PDX).

A primary focus of the Core is to develop, and provide to the BCM PDX community, computational and bioinformatics infrastructure to support large scale generation, characterization, and use of Mouse PDX and CAM-PDX models from a variety of organ sites, including breast, head and neck, pancreas, brain, etc. as well as other cancer types of interest to BCM investigators including pediatric cancers. The Core also provides expertise in transplantation and animal handling and assists with the evaluation of experimental therapeutics using PDX and CAM-PDX in vivo platforms in conjunction with those investigators maintaining PDX collections for each organ/disease type.

Mouse-PDX work is conducted in dedicated housing and surgical suites in the Transgenic Mouse Facility of BCM, a fully AAALAC accredited animal care and housing facility. Work is supported by the Center for Comparative Medicine which administers the facility.

CAM-PDX work is conducted in dedicated space in the Neurosensory Tower.

SERVICESMouse-PDX:

Development of computational/bioinformatic infrastructure to support PDX-based research.

Assist with, or facilitate, the generation of PDX models.

Facilitate in vivo treatment experiments with investigational drugs with PDX models or cell lines

Training for PDX related procedures. Coordinate provision of snap frozen tissue, viably frozen tissue, serum/plasma, and FFPE blocks/slides from PDX models from PDX program leads.

Coordinate provision of molecular derivatives of PDX models from PDX program leads.

Provide excess immunocompromised SCID/Bg mice from our breeding colony to BCM investigators.

PATIENT-DERIVED XENOGRAFT AND ADVANCED IN VIVO MODELS CORE (NEW 2017)

Michael Lewis, PhD

Academic Director

Mouse-PDX Associate Professor Breast Center

713.798.3296

[email protected]

CORE LEADERSHIP

Lacey Dobrolecki, MS

Core Director

Mouse-PDX Research Associate Breast Center

713.798.9024

[email protected]

Andrew Sikora, MD, PhD

Academic Director

CAM-PDX Associate Professor Otolaryngology

713.798.3909

[email protected]

Ravi Pathak, PhD

Core Director

CAM-PDX Instructor, Otolaryngology

713.798.8609

[email protected]


Tenascin C and Integrin Alpha 9 Mediate Prostate Cancer Interactions with Bone (Figure 2)

Molecular Predictors of Differential Chemotherary Response in Mouse Breast Cancer-PDX Models (Figure 1)

CAM-PDX: Conversion of cancer cell lines into 3D vascularized tumors Establish Patient Derived Xenografts (PDX) on the chicken egg chorioallantoic membrane (CAM-PDX)

Custom bioassays including angiogenesis and metastasis Drug sensitivity screening on 3D vascularized tumors and PDX Assistance in end-point assays (Flow cytometry, DNA/RNA purification, IHC)

Mouse-PDX Models Available 51 Breast Cancer Models 8 Pancreatic Cancer Models

Rebeca San Martin, Ravi Pathak, Jain Antrix, Sung Yun Jung, Susan G. Hilsenbeck, María Cristina Piña Barba, Andrew Sikora, Kenneth J. Pienta and David R. Rowley. (unpublished date)


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The Population Sciences Biorepository (PSB) serves as a resource for centralized cost-effective biospecimen processing and storage for epidemiological, translational, and clinical studies. The PSB also provides risk factor and clinical data collection. Services are available for individually funded investigators as well as for clinical centers that require prospective banking of specimens from patients for future research projects. The PSB team will consult with you to plan for data collection and specimen processing and storage needs for your projects. In addition, the PSB has a banked collection of annotated samples from a variety of cancer types that are available for individual investigator use. Contact the PSB to learn how to gain access to these important samples.

MAJOR EQUIPMENT CryoBioSystem MAPI high-security straw system QIAcube robotic workstation Chemagic Prepito-D extraction system Perkin Elmer Janus automated workstation Nano-drop 1000 MVE 1536P LN2 vapor freezers VWR -80°C mechanical freezers Thermo Scientific VisionMate scanner Thermo Scientific 8-channel decapper Barcode printers and scanner system

SERVICES Patient consenting, phlebotomy, and data collection Questionnaire development and administration Full fractionation and aliquoting for blood and urine samples DNA extraction from whole blood, buffy coat, plasma, or saliva RNA extraction from whole blood or buffy coat DNA Quantitation (absorbance and pico-green fluorescence) Whole Genome Amplification Long-term specimen archival

POPULATION SCIENCES BIOREPOSITORY (PSB)

Anne Truong

Assistant Laboratory Director

713.798.7480

[email protected]

CORE LEADERSHIP

Michael Scheurer, PhD

Academic Director

Associate Professor, Department of Pediatrics, Section of Hematology-Oncology

713.798.5547

[email protected]



Family-based exome-wide assessment of maternal genetic effects on susceptibility to childhood B-cell acute lymphoblastic leukemia in Hispanics.

A family-based exome-wide association study of maternal genetic effects was conducted among Hispanics with childhood B-cell ALL. A discovery cohort of 312 Guatemalan and Hispanic American families and an independent replication cohort of 152 Hispanic American families were used. Three maternal single-nucleotide polymorphisms (SNPs) approached the study threshold for significance after correction for multiple testing (P<1.0�×�10−6): MTL5 rs12365708 (testis expressed metallothionein-like protein [tesmin]) (relative risk [RR], 2.62; 95% confidence interval [95% CI], 1.61-4.27 [P�=�1.8�×�10−5]); ALKBH1 rs6494 (AlkB homolog 1, histone H2A dioxygenase) (RR, 3.77; 95% CI, 1.84-7.74 [P�=�3.7�×�10−5]); and NEUROG3 rs4536103 (neurogenin 3) (RR, 1.75; 95% CI, 1.30-2.37 [P�=�1.2�×�10−4]). This first family-based genome-wide association study for childhood ALL identified 3 maternal SNPs that may play a modest role in susceptibility for the commonest malignancy among Hispanic children.

Archer NP, Perez-Andreu V, Scheurer ME, Rabin KR, Peckham-Gregory EC, Plon SE, Zabriskie RC, De Alarcon PA, Fernandez KS, Najera CR, Yang JJ, Antillon-Klussmann F, Lupo PJ. Family-based exome-wide assessment of maternal genetic effects on susceptibility to childhood B-cell acute lymphoblastic leukemia in Hispanics. Cancer. 2016 Dec 1;122(23):3697-3704.

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The goal of this Core is to provide investigators with high quality mouse monoclonal antibodies (MAbs) and purified recombinant proteins to facilitate their research programs. The Core has experience with synthetic peptides, intact proteins and subcellular fractions as antigens and in generating MAbs that perform for a range of applications including immunoblotting, immunocytochemistry, immunoprecipitation, ELISA, and detection of phosphorylation sites. Expression and purification of recombinant proteins from either E. coli, mammalian cells including HEK 293 and CHO, and baculovirus infected insect cells compliments MAb services as a source of protein antigen. Protein expression and purification services also provide recombinant proteins for biochemical experimentation, as targets for drug screening and for structure analysis.

MAJOR EQUIPMENT ClonaCell EasyPick Robotic Colony Picking Instrument (StemCell Technologies) for cloning and screening of hybridomas directly from fusions

Bio-Rad BioLogic LP Chromatography System for antibody purification

Nexcelom Automated T4 Cell Counter Forma Stericult CO2 incubators for hybridomas BOD (27°C) incubators for insect cell cultures Hollow fiber bioreactors (FiberCell) for mass production of monoclonal antibodies in vitro

Spinner vessels(150 ml - 500 ml) for protein production in insect cells

Stirred tank bioreactors (5L) for large scale protein production in insect cell cultures

Akta Pure FPLC chromatography system with extensive collection of chromatography columns.

Microfluidics LM20 cell disruptor for processing large volume cultures.

SERVICES Generation of mouse monoclonal antibodies (MAbs) using hybridoma technologies. Includes immunization of mice, cell fusions, growth, screening and cloning of hybridomas, and cryopreservation of selected positive clones.

Production and purification of monoclonal antibodies from large scale hybridoma cultures (10mg - 1g amounts).

Generation of recombinant baculoviruses for protein expression in insect cells. Isolation of recombinant virus from cell transfections, plaque purification, and titering of viral stocks.

Production and purification of recombinant proteins from baculovirus vectors in large scale insect cell cultures, mammalian cell lines such as HEK and CHO cells, and from large scale E.coli culture.

Analysis and Q/C of purified protein products.

PROTEIN AND MONOCLONAL ANTIBODY PRODUCTION (RE-ORGANIZED 2017)

Dean P. Edwards, PhD

Academic Director

Professor, Department of Molecular & Cellular Biology and Pathology & Immunology

713.798.2326

[email protected]

Kevin Phillips, PhD

Academic Co-Director

Assistant Professor, Department of Molecular & Cellular Biology

713.798.6946

[email protected]

Kurt Christensen, BS

Lab Manager, Department of Molecular & Cellular Biology

713.798.2325

[email protected]

CORE LEADERSHIP


Figure 1. Development and characterization of a TDRD1 mouse monoclonal antibody. (A) Primary structure of human TDRD1 protein, which contains four tudor domains and one MYND domain with unknown function. (B) A core-generated mouse monoclonal antibody (#1277) specifically recognized the full length TDRD1 protein (130 kDa) in VCaP cells. The VCaP cells were transfected with control siRNA or two individual TDRD1 siRNA for 72 hr (Stealth, Invitrogen). (C) Expression of TDRD1 and ERG in commonly used prostate cancer cell lines, including LNCaP, LAPC4, VCaP, 22RV1, PC3, and DU145. (D) Interdependence of TDRD1 and ERG proteins levels in VCaP cells. VCaP cells were transiently transfected by individual siRNA against ERG or TDRD1 for 72 hr. (E) The half-lives of TDRD1, ERG, and AR proteins were determined by cycloheximide (CHX) chase assay in VCaP cells.

Figure 2. Detection of TDRD1 by immunohistochemistry (IHC) of FFPE tissue sections with mouse monoclonal antibody (#1277). No staining of normal human prostate that lack expression of TDRD1 (A), and specific cytoplasmic staining of prostate cancer known to express TDRD1 (B). A consecutive prostate cancer section was stained with a rabbit monoclonal ERG antibody (EPR3864) and revealed strong staining in both nucleus and cytoplasm in the cancer cells (C). The insets in (B) and (C) show the magnified images.


Monoclonal Antibodies to TDRD1, a Novel Biomarker for Human Prostate Cancer

Understanding the activation mechanism of cGMP-dependent protein kinase by X-ray crystallography - Choel Kim, Ph.D., Department of Pharmacology

The Core constructed recombinant baculovirus vectors for fragments of cGMP-Dependent Protein Kinase 1 (PKG1) and expressed the proteins in Sf9 insect cells. Five liter bioreactors were used for the production with yields from 5-10 mg of purified protein/bioreactor. Using these expressed PKG1 domain proteins, Dr. Kim and his lab were able to generate crystals from the C-domain and determine high resolution structure at 1.83 Å for the first time

Overall structure of the PKG I catalytic domain-AMPPNP complex. Domain organization of PKG I – the catalytic domain fragment used for the crystallization is shaded in gray (A). (unpublished).

Xiao L, Lanz RB, Frolov A, Castro PD, Zhang Z, Dong B, Xue W, Jung SY, Lydon JP, Edwards DP, Mancini MA, Feng Q, Ittmann MM, He B. The germ cell gene TDRD1 as an ERG target gene and a novel prostate cancer biomarker. The Prostate. 2016 76(14):1271-1284

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The Core performs non-radioactive RNA in situ hybridization (ISH) on tissue sections. A unique high-throughput technology developed by the Core (Yaylaoglu MB, Titmus A, Visel A, Alvarez- Bolado G, Thaller C, Eichele G. Dev Dyn. 2005 Oct;234(2):371-86) is used to determine gene expression patterns on sections, with an emphasis on tissues from rodent experimental models. The Core provides a full service that includes collecting, of animal tissue specimens, preparation of frozen sections, preparation of RNA probes from customer templates, conducting high-throughput ISH and documentation and quantification of expression patterns by microscopy. For human studies, customers must provide tissue sections.

MAJOR EQUIPMENT Tecan EVO Genepaint robot (for automated RNA in situ hybridization)

Three cryostats (Leica) Autostainer (Leica) Automated coverslipper (Leica) Zeiss Axio Scan.Z1 (brightfield and fluorescence)

SERVICES RNA in situ hybridization on tissue sections–brightfield or fluorescence development

Tissue processing and embedding (frozen tissue) Sectioning (cryostat) Preparation of non-radioactive RNA in situ probes (DIG- or FITC labeled)

X-gal staining (sections) Imaging (automated mosaic images) Automated quantification of in situ hybridization signals, brightfield only (gene expression levels and cell counts)

RNA IN SITU HYBRIDIZATION


Cecilia Ljungberg, PhD

Academic Director

Assistant Professor Department of Pediatrics – Neurology

832.824.8873

[email protected]


Disruption of Circadian Homeostasis Induces Leptin Resistance in WT Mice.

A) Summary of pSTAT3 signals detected by immunohistochemistry (IHC) (left) and POMC signal detected by in situ (right) in ARC of control WT, Cry1-/-;Cry2-/-, and Per1-/-;Per2-/- mice and jet-lagged WT mice, with signals detected in the ARC of control WT mice at ZT2 as arbitrary unit 1 (±SEM). B) Representative POMC in situ images in ARC of mice described in (A).

Kettner NM, Mayo SA, Hua J, Lee C, Moore DD, Fu L. Circadian Dysfunction Induces Leptin Resistance in Mice. Cell Metabolism 2015, 22:448-459. PMID: 26166747. PMCID: PMC4558341

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The goal of the Single Cell Genomics Core (SCGC) is to provide comprehensive services for DNA and RNA profiling on single or a small number of cells. The core is equipped with the C1 auto cell prep, HD Biomark, Access Array platform, and iCELL8 single cell system. Combination of these platforms allows cost-effective and high throughput DNA and RNA profiling ranging from a set of genes to the entire genome.

MAJOR EQUIPMENT Fluidigm Access Array: A fully integrated system that enables amplicon tagging and long-range PCR on Access Array IFCs for genomic regional capture and library preparation for next-generation sequencing.

Fluidigm BioMark HD System: A fully integrated real-time PCR system that enables analysis of gene expression, genotyping, mutant detection, and absolute quantification of nuclei acid sequences.

C1 Single Cell Auto Prep: The C1 Single Cell Auto Prep enables the isolation and preparation of individual cells for single-cell gene expression analysis in a streamlined fashion.

Wafergen iCELL8 system: A MultiSample NanoDispenser (MSND) system that is capable to isolate up to 1,800 cells with mix types and sizes on each chip for RNA-Seq.

SERVICES

Single cell capture and cDNA synthesis: Provide service for capturing from 100-1,600 cells per experiment, isolating mRNA, performing cDNA synthesis and sequencing library preparation from each individual cell.

Single cell capture and genomic sequencing: Provide service for capturing up to 800 cells, isolating genomic DNA, and prepare NGS sequencing library for each individual cell per experiment

High-throughput targeted gene expression assay: Measuring expression level of a set of genes using the HD Biomark platform, up to 96 samples times 96 genes per run.

High-throughput SNP genotyping: Up to 96 SNP genotyping for 96 samples per run using the HD Biomark platform.


Rui Chen, PhD

Academic Director

Associate Professor, Molecular & Human Genetics

713-798-5194

[email protected]

SINGLE CELL GENOMICS CORE (NEW 2017)



Comparison between single cell and population RNA sequencing

Zhang K, Gao M, Chong Z, Li Y, Han X, Chen R, Qin L. Single-cell isolation by a modular single-cell pipette for RNA-sequencing.Lab Chip. 2016 Nov 29;16(24):4742-4748.

Correlations between single cells and bulk cells in RNA-seq measurement of gene expression with Pearson correlation coefficient (r). (A) Single-cell 1 versus single-cell 2. (B) Single-cell 1 versus bulk cells. (C) Single-cell 2 versus bulk cells. Relative gene expression was estimated by calculating fragments per kilobase transcript per million mapped reads (RPKM).

Single cell RNA sequencing identifies three distinct cell types in the tissue

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The goal of this Core is to provide investigators with access to high quality in vivo and in vitro magnetic resonance imaging (MRI) for imaging and spectroscopy to accommodate their research projects. MRI offers unprecedented in vivo and longitudinal access to anatomical and physiological processes. Molecular imaging allows access to imaging some sub-cellular processes. Additionally, it is possible in many scenarios to image migrating labeled cells with MRI. Additionally, high resolution scans on the order of 20 microns can also be achieved in fixed samples ranging from mouse embryos to white matter tractography in pig brains. MRI is an outstanding resource for both in vivo and in vitro phenotyping.

MAJOR EQUIPMENT 9.4T, 20 cm bore, Bruker Biospin Avance I imaging system 1H volume and surface coil for rat, mouse, chicken and mouse embryos and fixed samples

Heteronuclear coils are also available. Workstations for image processing.

SERVICES Pristine in vivo anatomical assessments Cerebral blood flow Diffusion tensor imaging (DTI) for white matter tractography Perfusion imaging 31P spectroscopy of metabolites 1H spectroscopy of metabolites MRI contrast agent assessments Diffusion imaging Amyloid beta plaque imaging Longitudinal tumor volume assessments Angiography Muscle imaging Magnetization transfer contrast (MTC) to assess white matter damage

In Utero Imaging in Rats and Mice CEST Imaging — Chemical Exchange Saturation Transfer Cardiac Imaging — EDV, ESV and EF Assessments Cardiac Imaging — Anatomical Assessments Cardiac Imaging — Stress/Strain Calculations Cardiac Imaging — In vivo Ca2+ influx changes in Myocardium

Fat Assessment 19F MRI — Imaging Inflammation 19F MRI — Imaging tagged cells (e.g. lymphocytes) Dynamic Contrast Enhancement (BBB. tumor and placenta permeability)

Resting state functional MRI in the rat and mouse brain

SMALL ANIMAL MRI (NEW 2017)

Robia G. Pautler, PhD

Academic Director

Associate Professor, Department of Molecular Physiology and Biophysics

713.798.3892

[email protected]



Top row, left to right — Mouse Brain, In vivo Chicken Embryo, gated image of the mouse heart.

Bottom row, left to right — Mouse brain with hydrocephalus, in utero, in vivo mouse embryo image, mouse adrenal tumor images


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Antibody-Based ProteomicsOne Baylor Plaza, Houston, TX 77030Alkek Building for Biomedical Research, Room R507

[email protected]

BioEngineering CoreOne Baylor Plaza, Houston, TX 77030 Taub Research Building, Room T130

[email protected]

Biostatistics and InformaticsOne Baylor Plaza, Houston, TX 77030 Cullen Building, 3rd and 4th Floors

[email protected]

Core for Advanced MR Imaging (CAMRI)One Baylor Plaza, Houston, TX 77030Smith Building, Room S104AF

[email protected]

Cell Based Assay Screening Services (C-BASS)One Baylor Plaza, Houston, TX 77030Taub Research Building Room T328

[email protected]

Cytometry and Cell SortingOne Baylor Plaza, Houston, TX 77030Taub Tower of Main Campus, T103 & T105DeBakey Building of Main Campus, M901.3

[email protected]

Drug DiscoveryOne Baylor Plaza, Houston, TX 77030Michael E. DeBakey Center, Room M904G

713.798.8132/1279

Genomic and RNA Profiling (GARP)One Baylor Plaza, Houston, TX 77030Anderson Building, Lab 325E, Office 333E

[email protected]

Genetically Engineered Mouse (GEM)One Baylor Plaza, Houston, TX 77030Margaret Alkek Biomedical Research Building, R223

[email protected]

Gene VectorOne Baylor Plaza, Houston, TX 77030Margaret Alkek Biomedical Research Building, R650

[email protected]

Human Tissue Acquisition and Pathology (HTAP)One Baylor Plaza, Houston, TX 77030ABBR Histology Service, R536; Immunochemistry.R531D

[email protected]

Human Stem Cell (HSCC)One Baylor Plaza, Houston, TX 77030 Ben Taub Research Center, Suite T143

[email protected] Office | 713.798.8211 Lab

Integrated MicroscopyOne Baylor Plaza, Houston, TX 77030Cullen Building Room 123A

[email protected]

Mass Spectrometry ProteomicsOne Baylor Plaza, Houston, TX 77030Jones Wing, Alkek Center for Molecular Discovery Rooms 108CB, 112C & 113C

[email protected]

CORE DIRECTORY


MetabolomicsOne Baylor Plaza, Houston, TX 77030Jones Wing, Alkek Center for Molecular Discovery Rooms 109C & 112C

[email protected]

MHC TetramerOne Baylor Plaza, Houston, TX 77030Smith Building, Room S230

[email protected]

Mouse Embryonic Stem Cells (ES)One Baylor Plaza, Houston, TX 77030Alkek Building for Biomedical Research Room R.761

[email protected] 713.798.1778

Mouse Metabolism CoreOne Baylor Plaza, Houston, TX 77030Alkek Building for Biomedical Research Room R618

[email protected]

Mouse PhenotypingOne Baylor Plaza, Houston, TX 77030Taub Animal Facility Room T001.T002

[email protected]

Optical Imaging and Vital Microscopy (OIVM)One Baylor Plaza, Houston, TX 77030Cullen Building, Rooms 112A & 114A

[email protected]

Patient-Derived Xenograft CoreOne Baylor Plaza, Houston, TX 77030 Mouse-PDX — Albert B. Alkek Building, Room N1210

[email protected]

CAM-PDX — Neurosensory Building, Room NA516

[email protected]

Population Sciences BiorepositoryOne Baylor Plaza, Houston, TX 77030Alkek Building for Biomedical Research, Room R531E

[email protected]

Protein & Monoclonal Antibody ProductionOne Baylor Plaza, Houston, TX 77030Alkek Building for Biomedical Research, Room R551E

[email protected]

RNA in Situ Hybridization1250 Moursund Street, Houston, TX 77030Jan and Dan Duncan Neurological Research Institute Suite N1325

[email protected]

Single Cell Genomics One Baylor Plaza, Houston, TX 77030Albert B. Alkek Building Room N1409

[email protected] 713-798-5194

Small Animal MRIOne Baylor Plaza, Houston, TX 77030M.D. Anderson Hall Room 103G

[email protected]

To learn more about Core labs, download a free QR code app on your SmartPhone and then scan this code.

www.bcm.edu/corelabs

[email protected]

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ADVANCEDTECHNOLOGY

C O R E SGC76457

ADVANCED TECHNOLOGY CORES CATALOG€¦ · I am pleased to present the BCM Advanced Technology Cores...

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Transcript of ADVANCED TECHNOLOGY CORES CATALOG€¦ · I am pleased to present the BCM Advanced Technology Cores...