1 -Animal house
The IGB animal house laboratory is a conventional structure of more than 600 square meters located in Via P. Castellino 111, Naples, Italy, authorized by the Ministry of Health according to the Italian law on the animal experimentation (D.L. 116/92).
The structure can house about 6000 animals, in particular rodent species like mice, rats, guinea pigs and rabbits. It is organized in many functional areas including those for quarantine and animal maintenance, a laboratory for surgical operation, a room for post-surgical permanence, washing and technical areas, offices. An independent conditioned and ventilation air system allows maintaining constant temperature (22°C) and humidity (55%) with twenty air changes each hour. Automatic controlled lighting guarantees the day/night biological cycle, with light from 8.00 am to 20.00 pm and dark for the remaining time. The access to the locals is filtered and clean-passages and corridors to reach the operational rooms have been designed. The organization of the facility, together with the appropriate procedures practised by the working staff, guarantees a perfect health status of animals in agreement with Specific Pathogen Free conditions, ensuring both the integrity of the barriers and health status.
The maintenance of the mouse facility unit is undertaken by a team of well-trained, highly professional and experienced personnel including: one technician for washing services, six animal husbandry technicians, two facility managers, one veterinarian healthcare consultant, one scientific Director.
The IGB animal house is open to the institutions of the Biotechnology Area NA1 such as the Institute of Genetics and Biophysics "Adriano Buzzati Traverso" (CNR), the Institute of Protein Biochemistry (IBP) and the Telethon Institute of Genetics and Medicine (TIGEM) but other scientific institutions as well as university investigators are allowed to access.
2 -Automation Laboratory (50 sqm). Stem Cell Fate Lab.(www.igb.cnr.it/scfl/)
Cell Maker, Robotic platform for HTS-Cellomics (Hamilton).
The innovative automation unit assembled, that was named Cell-Maker, allows standardization of time-consuming tasks as microplate coating, cell plating, washing, media changes, compound addition and multi-signal reading (fluorescence, chemo-luminescence and absorbance). Its configuration, including hardware (pipetting unit and integrated instrumentation) and software components, was adapted to properly handle ES cells and efficiently drive their differentiation.
- HTS screening
The SCF Lab provides a high-throughput screening facility based on our automated system for murine ES cell differentiation, the Cell-Maker. The screening platform is able to process up to 4000 samples simultaneously in a 96-well microplate format and is adapted to the demands of multiple cell-based functional assays (readouts: absorbance, fluorescence, luminescence). In addition, a variety of biochemical assays can also be performed, depending on the specific requirements. The compound collections or chemical libraries supplied by demanders (single compounds, mixtures or combinatorial pools) should be provided in 96-well microplates. All screening data are automatically processed to test quality-control parameters (as Z" factor). Reporting data options: database exports (Excel or Word files), Excel tables and graphical views. To be confident of the quality of your hits, we retest all putative hits by multi-well hit confirmation experiments.
-Cellometer Auto T4/Nexelom Bioscience)
-Nucleofector Device (Amaxa Biosystems)
The Nucleofector technology developed by Amaxa is a non-viral transfection method for a wide range of difficult-to-transfect cell lines and primary cells. Using this technology, DNA is delivered straight into the nucleus ensuring higher transfection efficiencies compared to other classical methods (i.e. electroporation).
3 -Conference Rooms
Equipped with complete audio-visual and videoconferencing systems
Accomodating max 200 persons
4 -Data Elaboration Centre
The mission of the Informatics (IT) Core is to provide an integrated system of computer-related products and Information Technology services, guaranteeing a constant and effective support for their use within IGB research programs. These services are aimed to fulfill and meet the needs of internal and external users (researchers and administration personnel), in terms of utility, quality, reliability and cost effectiveness. Specifically, the objecives assigned to the IT Core team include the following ones:
-to guarantee the efficient operation and the regular maintenance, under a centralized management, of the IGB information system (including both infrastructures and services);
-to organize and manage IGB data networks, ensuring their connection to the internet webnet;
-to organize and support all the IT-related operational procedures for IGB, in terms of design, implementation, management and update of such procedures;
-to organize and manage the installed IT equipment at IGB;
-to develop new information services and applications for students, researchers and administration personnel;
-to ensure assistance and consulting services for teaching and scientific activities at IGB;
-to promote and provide training on topics related to informatics, communication and telematic services;
- to promote, and participate to, expertimental projects in the areas of research in collaboration with other units at IGB;
-to define, in the context of the general mission of IGB, standards and rules to guarantee appropriate levels of information security and interoperability for all the units of IGB.
-Centralized printing system
-Institutional web site
Projects in progress:
-Dedicated file servers for each group - "group areas"
-Remote Authentication for network access
-Idisk (virtual disk with webdav protocol)
-Shared online calendars
5 -Cell Culture Laboratories
All equipment needed for the growth, maintenance an analysis of cell.
-Flow hoods (n. 15)
-Atmosphere controlled incubators (n.20)
-Phase contrast microscopes
-Freezer - 80°C (n. 20)
6- Functional Genomics Service
Real Time PCR. Real Time PCR technology is based on the correlation "in real time" between the fluorescence emitted by a fluorochrome bound to a biological sample during the PCR reaction, and the starting amount of DNA template; this technology is utilized especially for quantitative analysis. Among the techniques utilized to develop a Real Time protocol, SYBR-green and TaqMan technologies are the most commonly used, the SYBR-green technology being the easier and more economical approach with respect to TaqMan technology that is highly specific, but with higher initial costs. Data from Real Time PCR analysis may be utilized in two different ways:
1) in an absolute way, with respect to a standard curve obtained with samples at known concentration (e.g. to perform studies on the viral charge, and/or microorganisms/OGM in a biological sample).
2) in a relative way, with respect to a normalization gene, to quantify gene expression.
We are equipped with three Real Time PCR apparatuses:
1) MJ Opticon II apparatus, with a 96 well cartridge and the capacity to operate in gradient .
2) Bio-Rad apparatus, with a 96 well cartridge and the capacity to operate in gradient.
3) Applera 7900HT Fast Real Time PCR. It is equipped with both 96 and 384 well cartridges: it can be used in FAST modality with the 96 well cartridge. Given specific experimental needs, it may happen that 384 and 96 wells cartridges will be used in different days.
- Expression Profiling. Expression Profiling analyses are performed using one-channel array technology. The facility has a complete Affymetrix GeneChip System, that exploits photolithographic/chemical coupled synthesis of oligonucleotides on silica wafer. The Genechip Affymetrix system includes hybridisation oven, washing station, and the new 7G scanner, allowing the processing of the full Affymetrix catalogue and custom design arrays. A dedicated PC station manages the array image and intensity analysis. This system can be employed for a growing number of applications including:
-Genome-wide expression profiles with the expression arrays.
-Gene regulation analysing as Protein/DNA interactions and ChIP on Chip with tiling arrays.
-Linkage analysis, association, and copy number studies with mapping arrays that contain thousands of SNPs.
The lab gives a full training for scientific and technical support and supervision in order to perform experiments with the Affymetrix system. Researchers provide all the reagents needed for the experiment, the labelled target to be hybridized on the specific experimental arrays, and their specific samples. The lab performs the whole process returning the RAW data obtained after conversion of the image file (DAT file) in a numeric file (CEL file). DAT files contain signal intensity for each hybridization where each signal intensity is converted into a numeric value
-High Throughput Sequencing. New DNA sequencing platforms deliver several orders of magnitude more sequence than is possible with the traditional methods, opening up new possibilities for the analysis of genomes, transcriptomes as well as small RNA populations. The deep sequencing methods may reveal novel insights about gene regulation, genome structure, and genome evolution.
The IGB recently acquired a SOLiD(TM) 3 System (Applied Biosystem) for high throughput sequencing. The facility space meets strict climatization standards necessary for the sequencer operations, and is made of multiple rooms to avoid cross-contaminations during the various steps of sample processing.
The SOLiD(TM) 3 System is based on massive parallel sequencing in short reads (25-70 bases) of pre-amplified, immobilized DNA template molecules, representing the whole genetic content of selected samples (DNA, cDNA from RNA, ChIPs, miRNAs, etc.). The short reads are aligned to a reference genome: the higher the number of reads, the "deeper" the analysis, the more robust the final outcome. The system uses an emulsion-based method to clonally amplify template fragments in vitro.
The system has a very high throughput capacity, generating up to 15 gigabases of data per slide. The accuracy greater than 99.94%, is due to 2 base encoding: the SOLiD(TM) System distinguishes itself by providing data that is significantly more accurate than alternative next-generation platforms for variation detection.
SOLiD(TM) 3 System supports sample preparation for mate-paired libraries with insert sizes ranging from 600 bp up to 10 kbp. This broad range of insert sizes combined with ultra high throughput and flexible two flow cell configuration enables more precise characterization of structural variation across the genome.
The several applications stemming from this technology range into different directions including metagenomic and de novo applications, providing definitive and whole-genome coverage of the information of interest, both at a quantitative and qualitative level.
The lab is fully equipped to provide researchers full support starting from individual samples with experimental setup, wet-lab experiments up to sample processing, running, and data acquisition.
7 -Integrated Microscopy
In the post-genomic era both light and electron microscopy have acquired a key role in advancing knowledge in the context of various fields of science (biology, development, immunology, genetics, cell and molecular biology). Indeed, modern microscopy techniques (confocal microscopy, live cell imaging, immuno-electron microscopy) allow the study of the static structure of biological systems, a prerequisite for understanding the function, as well as providing the possibility of a dynamic study of biological processes such as signaling, excitability, transport, apoptosis and development.
A fee-for-service integrated microscopy facility was created at IGB to provide:
-management and use of high complexity equipments;
-continuous support and guidance for all research groups;
-technical support to preparation of investigators' samples, imaging and data analysis;
-training to undergraduate and PhD students, post-docs, researchers;
-protocol design assistance;
-set up and application of new microscopy technologies;
-collaborations with other CNR Institutes, Universities, or companies (public or private).
The facility is located on the first floor of the Building 3 in the Pietro Castellino Research Area. Equipment and staff are available to everyone within the IGB.
Researchers from other institutes in the area or universities or companies will be equally welcome. The usage of the scientific common resources of the IGB services by outside laboratories must be authorized (see convention by the Statute and the Rules).
Flow cytometry is a technology that measures and analyzes the morphological and optical characteristics of single cells suspended in a stream of saline fluid. A focused beam of laser light illuminates each moving cell and light is scattered in all directions. Detectors placed forward of the intersection point or side-on (with respect to the laser beam) receive pulses of scattered light that are converted into a form suitable for computer analysis and interpretation. The physical (morphological) profile of a cell can be observed by combining forward light scatter (FSC) and side light scatter (SSC) analysis. The laser can excite fluorophores that have been used to mark various molecules or physiological functions of the cells. The use of fluorophores with different fluorescence characteristics allows simultaneous multiparametric analysis. The combination of scattered and fluorescence generate useful data to analyze heterogeneous cell populations.
IGB-ABT FACS Core
The IGB flow cytometry is based on Becton Dickinson FACSAria. This instrument can register up to 70000 events/second and this guarantees a good signal linearity. It is equipped with 2 lasers beam: blue (488 nm) and red (633nm) that detect 8 different emission signals of fluorescence for a multiparametric analysis. The instrument can contemporarily separate 4 cellular populations by analyzing events and dividing them at a maximum speed of 3000 cells/second. To separate cells it uses a dropping device that guarantees appropriate temperature, sterility and high cell recovery.
The service provides:
1)Technical support in terms of protocol design assistance and data analysis required by the investigators.
2) Phenotypic analysis of mammalian cells:
-cell cycle analysis
-surface phenotypic cell analysis
-intracellular staining of fixed cells
-study of cell survival-apoptosis and cell proliferation.
3) Sorting of mouse and human cells heterogeneous single cell populations into homogeneous populations.
4) Sorting of fluorescent transfected cell lines