ANIMAL MODELS PLATFORM

REFERENT SCIENTIST Prof. Adriana Maggi – Animal Engineering Lab: Director of the Center of Excellence on Neurodegenerative Diseases, Università degli Studi di Milano (adriana.maggi@unimi.it)

TO ORDER/FOR INFORMATION

services@fondazionefilarete.com

INTRODUCTION

The platform aims to offer its services to basic research groups and pharmaceutical companies for the study of drug action in complex model systems and for the generation of models in which preclinical and clinical phases of drug research can proceed as a continuum thanks to the use of markers of drug action and methodologies (imaging) which can be used in small animals as well as in humans.

SERVICE FEES

ANIMAL MODEL SERVICES

Real Time PCR

7900 HT Fast Real Time PCR combines 96 and 384-well plate compatibility also offers optional Fast real-time PCR capability. Combined with TaqMan Genomic assays enable to achieve unprecedented throughput and flexibility, allowing to pursue projects beyond the scope of previous real-time instruments.

In their 96 well format these Instruments allow to run 96 samples in less than 2 hours. It is possible to update to 384 well plate format or to the Micro Fluid card format.

Taq Man Assay (assay design samples quality control, reaction, analyses)

Design Taq Man Assay (assay design samples quality control, reaction, analyses)

Micro Fluidic Card (assay design samples quality control, reaction, analyses)

cDNA RNA extraction

Retro-transcription

Maxi – Plasmid DNA Preparation Kit qiagen, Plasmid quality Preparation, high purification (transfection in eukaryotic cells 500 ug – 1 mg)

Mini – Plasmid DNA Preparation Kit qiagen, Plasmid quality Preparation

Competent Cells Competent cells bacterial strains : xl1blue (107) , dh5alpha (106) , HB101 (106), sure (105)

Transformation Transformation of plasmid DNA into bacteria cells : xl1blue , dh5alpha , HB101, sure

Qualitative/quantitavie RNA analysis with Experion automated electrophoresis system

Qualitative/quantitavie PROTEIN analysis with Experion automated electrophoresis system

Qualitative/quantitavie DNA analysis with Experion automated electrophoresis system

Spectrophotometer Tecan CM Sunrise™ (1 hour)

Spectrophotometer Nanodrop ND-1000 (1 hour)

Biorad Experion 700-7002 Automated Electrophoresis Station (1 hour)

Promega GloMax®-Multi Detection System (1 hour)

Upright Microscope Leica DM2500 (1 hour)

Manual Inverted Microscope Leica DMI3000 B (1 hour)

Real Time PCR rental (per hour)

* = for al least 10 items

PLATFORM CAPABILITIES

ANIMAL ENGINEERING PLATFORM

Modification of models of human diseases for the study of novel therapies by the use of imaging

Application of innovative animal models, such as reporter mice, to pharmacological research

Application to imaging technologies to the study of drug pharmacokinetics and pharmacodynamics

Generation of stem and neural reporter cells

Generation of innovative systems of immobilized reporter cells for drug functional screening

Generation and testing of innovative systems for drug delivery

A practical training in the field of Animal engineering and Imaging

Modelli animali (ANIMAL MODEL)

TITOLO (TITLE): IMAGING MOLECOLARE IN DRUG DISCOVERY E SVILUPPO PRE CLINICO (MOLECULAR IMAGING IN DRUG DISCOVERY AND PRE-CLINICAL DEVELOPMENT)

DURATA (DURATION): 16 ore (8 teoria – 8 pratica) (16 hrs – 8 theory 8 practice)

CONTENUTI (CONTENT):

L’imaging funzionale e molecolare nelle indagini biologiche, nella farmacologia e nella medicina. Gli strumenti e il supporto informatico necessari. Il drug discovery in oncologia e sistema nervoso centrale. Meccanismi predittivi di tossicità ed efficacia del farmaco nella sviluppo preclinico. Sezioni teoriche e pratiche.

(Functional imaging investigations and molecular biology, pharmacology and medicine. Tools and IT support required. The drug discovery in oncology and central nervous system. Predictive mechanisms of toxicity and efficacy in preclinical development. Theoretical and practical sections.)

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

REFERENCES Stell A., Et Al, Multimodality imaging: novel pharmacological applications of reporter systems. Q J Nucl Med Mol Imaging. 51, 127-38 (2007) Stell A., Et Al Cancer modeling: modern imaging application in a generation of novel animal system sto study cancer progression and therapy. Int. J. Biochem Cell Biol. 39, 1288 - 96 (2007) Maggi A., Salvatore M., Molecular Imaging. Q J Nucl Med Mol Imaging. 51, 95 (2007) Stell A., Belcredito S., Ramachandran B., Biserni A., Rando G., Ciana P., Maggi A. Multimodality imaging: novel pharmacological applications of reporter system. Q J Nucl Med Mol Imagin. 51, 127-38 (2007) Montani C., Penza M., Jeremic M., Biasiotto G., La Sala G., De Felici M., Ciana P., Maggi A., Di Lorenzo D. Genistein is an efficient estrogen in the whole-body throughout mouse development. Toxicol. Sci. 103, 57 – 67 (2008) Biserni A., Gianessi F., Sciarroni Af. Milazzo Fm., Maggi A., Ciana P. In vivo imaging reveals selective peroxisome proliferator activated receptor modulator activity of the synthetic ligand 3-(1-(4-chlorobenzyl -3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid (MK-886). Mol. Pharmacol. 73, 1434 – 43 (2008) Cignarella A., Bolego C., Pelosi V., Meda C., Krust A., Pinna C., Gaion R.M., Vegeto E., Maggi A. District roles of estrogen receptor-α and β in the modulation of vascular inducible NO synthase diabetes. J Pharmacol Exp Ther. Oct 2 (2008) Vegeto E., Benedusi V., Maggi A. Estrogen anti-inflammatory activity in brain: A therapeutic opportunità for menopause and neurodegenerative diseases. Frontiers in Neuroendocrinology 29, 507-19 (2008) Di Lorenzo D., Rando G., Ciana P., Maggi A. Molecular imaging, an innovative methodology for whole body profiling of endocrine disrupter action. Toxicological Sciences 106, 304 – 311 (2008) Stell A., Belcredito S., Ciana P., Maggi A. Molecular imaging provides novel insights on estrogen receptor activity in mouse brain. Molecular Imaging 7, 11 – 21 (2008) Amadasi A., Mozzarelli A., Meda C., Maggi A., Cozzini P. Identification of Xenoestrogens in food additives by an integrated in silico and in vitro approach. Chem. Res. Toxicol. 22, 52 – 63 (2009) Brufani M., et al. Novel Locally Active Estrogens Accelerate Cutaneous Wound Healing. A Preliminary Study. Molecular Pharmaceutics. 6, 543-556 (2009)

SPECTROPHOTOMETRY

Tecan CM Infinite® M 200 NanoQuant

The Infinite 200 NanoQuant is a multimode microplate reader developed specifically for absorbance applications with low sample volume This sensitive instrument is equipped with UV-stable filters, and can detect DNA concentrations as low as 1 ng/μl. The Infinite 200 NanoQuant can be used for a broad range of applications as for example measuring DNA- or RNA- quantification or quality control and labeling efficiency. It delivers excellent sensitivity, multiplexing capability and high format flexibility including 6 – 384-well microplates as well as half-area plates. In the NanoQuant plate a special quartz optic for each of the 16 samples guarantees outstanding performance and a high rate of reproducibility. The plate offers quick and easy cleaning to reduce the risk of cross contamination. In addition after a working day the plate can be even cleaned in an ultra sonic water bath for additional efficient cleaning. The NanoQuant plate is compatible with an eight-channel pipette to easy dispense the samples on the 16 positions of the tool.

Tecan CM Sunrise™

Sunrise is a versatile microplate absorbance reader for 96-well plates providing all the functionality needed for the numerous applications in diagnostics, biotechnology and research laboratories

· Supports ELISA- & kinetics-, as well as agglutination reading · Selection of user interface: Netbook* or Touchscreen · Selection of eight languages for easy operation · 3 year warranty as Tecan´s commitment to quality · Reads plate in only 6 sec. via advanced 12-channel optics · Powered by smart MagellanTM data analysis software · Quick access to your program favourite via start-screen · Advanced curve-fitting & plate-to-plate QC functions · IQ / OQ procedure & documentation · Designed to meet IVDD 98/79/EC & FDA´s 21 CFR part 11** · Filter 405, 450, 492 and 620 nm

A set of options such as temperature control up to 42°C, a gradient filter for wavelengths scanning and a barcode scanner allow to customize the Sunrise to your application needs. The compact space saving design makes the Sunrise ideal for stand-alone use, as well as integration into automated ELISA-systems such as the Freedom EVOlyzer®.

Nanodrop ND-1000

The NanoDrop ND-1000 is a full-spectrum spectrophotometer (UV and visible spectrum, 220-750 nm) for measuring the absorbance of DNA, RNA, proteins and dyes. The newly developed sample retention system allows for measurement of small samples volumes: the sample droplet is held in place by surface tension when it is slightly compressed between the pedestal and the sample arm; this generates the defined pathway of 1 mm. The spectrum measurement is then performed with two optical fibers installed in the pedestal (emitting light of a Xenon lamp) and the sample arm (spectrometer with linear CCD array). Quantification is performed based on the spectrum measurement at the defined pathway of 1 mm. DNA, RNA, protein or dye in 1 µl can be measured without using a cuvette or capillaries. The NanoDrop ND-1000 is a small apparatus (20 cm x 14 cm). A measurement only takes 10 sec. Sample measurements are possible over a wide range of concentrations, e.g. double-stranded (ds) DNA: 2-3700 ng/µl, single-stranded (ss) DNA: ≤ 2400 ng/µl, RNA: ≤3000 ng/µl, and protein: 0.10-100 mg/ml BSA (at 280 nm); this is 50x higher than ranges of normally used spectrophotometers. Once the measurement is complete,the surfaces are simply wiped witha lint-free lab wipe.

Beckman DU® 730 UV/Vis Scanning Spectrophotometer

The narrow bandwidth allows you to scan your samples with excellent resolution.

With its focused-beam design, the system provides optimal and reproducible results for small samples

REAL TIME PCR Applied Biosystems 7900 HT Fast Real-Time PCR System

Applied Biosystems 7500 Real-Time PCR System

Real-time Polymerase Chain Reaction (rtPCR or quantitative PCR - qPCR) is one of the most powerful and sensitive gene analysis techniques available. It is used for a broad range of applications including :

• quantitative gene expression analysis, • SNP analysis, • pathogen detection, • drug target validation and for measuring RNA interference. • Microarray Verification • Quality Control and Assay Validation • Copy Number Variation • MicroRNA Analysis • Viral Quantitation • siRNA/RNAi experiments

Adavantages in the use of Real-Time PCR versus traditional PCR include:

• Increased dynamic range of detection • No post-PCR processing • Detection is capable down to a 2-fold change • Data are collected in the exponential growth phase of PCR • The cleaved probe provides a permanent record amplification of an amplicon

MOLECULAR BIOLOGY IMAGING

Biorad Molecular Imager ChemiDoc XRS System

The ChemiDoc XRS+ system is the most advanced chemiluminescence detection system in the Bio-Rad gel documentation line. The system features flat fielding technology for superior uniformity and quantitation, and it has the flexibility to image chemiluminescent, fluorescent, and colorimetric samples. The system includes a supersensitive 16-bit CCD camera, light-tight compact darkroom, slide-out transilluminator with preparative mode for added safety when manually visualizing samples, software-driven motorized lens and illumination sources, and powerful, user-friendly Quantity One 1-D analysis software. Unlimited copies of Quantity One Basic software, available with each system, allow free functionality for sharing, analysis, and presentation of gel data.

The ChemiDoc XRS+ system can be used for imaging in a wide variety of applications (western blotting, nucleic acid detection, 2-D gel electrophoresis, dot blotting, densitometry, and colony counting) using a large portfolio of detection methods (Immun-Star HRP and AP chemiluminescence detection as well as ethidium bromide, SYBR Green I, SYPRO Ruby, Cy3, rhodamine, Green Fluorescent Protein, Hoechst, silver, copper, and Coomassie Blue staining).

Biorad Molecular Imager VersaDoc MP 4000 System

VersaDoc imaging systems are high-quality, flexible instruments that allow imaging of a wide range of samples — from 1-D and 2-D gels to chemiluminescent blots, microplates, and autoradiograms.

· A Wide Range of Supported Applications · Proteomic and genomic studies · Protein gels (2-D), including DIGE and other multichannel

differential electrophoresis techniques · Nucleic acid and protein gel imaging (1-D) · Multiplex gel and blot imaging · Western, northern, and Southern blot imaging · Quantum dot imaging · Colony counting

The standard configuration includes a supersensitive, deeply cooled CCD camera, darkroom, power supply, cables, epi-illumination (blue and white), transillumination (UV), fluorescent reference plate, focusing target, and Quantity One 1-D analysis software. Optional accessories include green and red excitation sources for increased multiplexing capabilities (useful for DIGE and imaging of quantum dots and organic fluroscent secondary antibodies). The VersaDoc MP 4000 system is an ideal imager for proteomic studies, offering maximum flexibility with the ability to image colorimetric and fluorescently stained gels and blots to detect chemiluminescent and other low-light and multiplex or multifluorescent samples. It resolves the finest details of every band or spot. Features include:

· 3.2 megapixel CCD with 53 µm resolution · Accurate and quantifiable collection of data with a CV ≤5% · Identification of differences in protein expression using optional PDQuest 2-D analysis

software

The camera is cooled to absolute temperatures to minimize background noise, enhance the signal-to-noise ratio, and maintain reproducible imaging conditions. This high-performance digital camera produces luminescent images of very faint samples (due to either low-abundance samples or faint signal) at speeds faster than exposure times of film-based detection. Features include:

· Supercooling to -35°C (absolute) for optimal imaging with low-light applications · True 16-bit data and a dynamic range that covers 5 orders of magnitude to maximize limit

of detection · Quantitation of differences in sample abundance using Quantity One 1-D analysis software · Red, green, blue, and UV illumination for multiplex quantitative imaging

Biorad Experion 700-7002 Automated Electrophoresis Station

The Experion automated electrophoresis station performs all of the steps of gel-based electrophoresis in one compact, durable unit. It automates analysis by combining electrophoresis, staining, destaining, band detection, and imaging into a single, 30 min step. It's an electrophoresis cell, power supply, and imager built into a single device and is designed for easy, precise operation:

· Highly accurate laser provides precise fluorescence detection · USB port allows easy installation and maximum usability · Built-in power supply reduces cost and saves bench space · The following types of kits are available for analysis of proteins, RNA, and DNA: · Experion starter kits · Experion protein analysis kits · Experion RNA analysis kits · Experion DNA analysis kits

The Experion automated electrophoresis system can be used upstream and downstream of a number of other nucleic acid- and protein-application focused products from Bio-Rad.

Promega GloMax®-Multi Detection System

The GloMaxR-Multi Detection System is an expandable multimode reader with unbeatable performance. Each detection mode has dedicated optics for the highest versatility without sacrificing performance. The GloMaxR-Multi Detection System can be used as a reader dedicated to a single mode or as a multimode reader. As your application needs expand, the system can easily accept the add-on modules, which offer additional detection modes. This flexibility allows you to customize the system to fit your laboratory needs.

Luminescence module · Sensitive to approximately 3 x 10-21 moles of luciferase · 9 log dynamic range · Ideal for cell-based assays · Engineered to minimize sample cross-talk · Direct-to-Excel® data importing: Requires PC to operate · Excel® macros allow simple data analysis for Dual-Luciferase® Assays

Fluorimeter module

FLUORESCENCE MICROSCOPY Upright Microscope Leica DM2500

• The Leica DM2500 microscope is the ultimate tool for demanding tasks in pathology, cytology, haematolgy, and even for basic research applications.

• With its powerful 100W illumination, high-quality optical performance, and state of the art accessories, the Leica DM2500 is especially well suited for challenging goals in pathology or biomedicine that require differential interference contrast or high-performance fluorescence. The bright 100W illumination is particularly beneficial for DIC work.

• The modular, application-oriented design means that the Leica DM2500 can be configured to fit specific applications and physical requirements of the user

Manual Inverted Microscope Leica DMI3000 B

• The Leica DMI3000 B research microscope supports all transmitted light methods including fluorescence, live cell, time-lapse imaging; high-speed multi-fluorescence optical sectioning, micromanipulation, and more.

• The viewing angle of the ergonomic tube, a standard feature, can be continuously adjusted to ensure the most comfort for the user.

• The Leica DMI3000 B’s viewing channel, a notch between the eyepieces, provides a clear view of the specimen regardless of the tube’s position. All of the microscope’s controls are easily accessible for convenient operation.

Automated Inverted Microscope Leica DMI4000 B

The Leica DMI4000 B automated inverted research microscope is ideal for scanning cell and tissue cultures. The system features a fluorescence axis for ultra brilliant fluorescence imaging.

• The internal filter wheel with motorized excitation manager and FIM (Fluorescence Intensity Manager) enables excitation of fluorochromes in less than 20 milliseconds.

• FIM regulates light intensity at five fixed levels and remembers the setting for each filter cube.

Inverted Microscope with LED Illumination Leica DM IL LED

• Samples are focused with the quadruple objective nosepiece.

• The transmitted light illumination allows the integration of the contrast slide (for phase and modulation contrast)

• The fluorescence filter cubes perform a matched combination of excitation, reflection, band-pass and barrier filters.

Stereomicroscope Leica M205 FA

Overview The Leica M205 FA combines the proven TripleBeam® technology with the unique FusionOptics™ concept to open up new dimensions in fluorescence stereomicroscopy.

• The fully apochromatically optics system, the largest zoom range available on the market, 20.5 : 1, and a resolution to 1050 lp / mm will provide you with a view of details hitherto unknown in stereomicroscopy.

• The Leica M205 FA allows time-intensive studies of living organisms and documentation of complex image series. Multiple fluorescence images are made possible and instantly reproducible with motorized focus, zoom, filter changer and iris diaphragm.

Key Features

• Never before seen: 3D images of highest resolution, brilliance and depth of field.

• Largest zoom range in stereomicroscopy.

• Brilliant fluorescence images, rich in detail and contrast

• Microscopes that grow with your requirements.

• Operating comfort and reproducibility through motorization.

• Intelligent control with SmartTouch™. • Encoding for reproducibility and

consistency in experiment procedures.

• Optimal display of your specimens with high-performance objectives.

• Solid foundation for your research: a stable mechanical construction.

• Integrated system solutions make life easier