Explore These Core Facilities
Bakhos A. Tannous, Ph.D.
The Neuroscience Center Vector Core was established to meet the increasing need of researchers for recombinant viral vectors capable of achieving efficient and long-lasting genetic modification of cells in primary cultures and living animals. Adeno-associated virus (AAV) and lentivirus vectors have emerged as the most efficient recombinant viral vectors to achieve these goals. Retrovirus vectors continue to be used in niche applications such as transduction of dividing progenitor cells for lineage analysis.
This Core offers advice to the investigators on AAV, lentivirus and retrovirus vectors most appropriate for their research needs based on relative infectivity of cells of interest, use for culture or in vivo studies, promoter considerations, hypotheses being addressed, and appropriate reporter genes. The Core provides appropriate plasmid backbones with different fluorescent markers and construction information and assistance, as well as packaging of vectors for the investigators. Investigators are responsible for inserting their genes of interest into the MCS of vector backbones, validating correct construction by restriction analysis, testing bioactivity by transfection assays (when possible), and supplying clean maxi-prepped DMA for packaging
For more information, contact:
Director of MassGeneral Vector Core
Bakhos A. Tannous, PhD
Department of Neurology
Molecular Neurogenetics Unit
149 13th Street, room 6309
Tel: (617) 726-6026
Fax: (617) 724-1537
Quantitative Real-Time PCR Core
Bakhos A. Tannous, Ph.D.
This Core offers quantitative measurement of nucleic acid (DNA or RNA) levels using real-time PCR. This includes Equipment and software for transcript quantitation, gene expression analysis, PCR melting curves, SNP genotyping, whole-genome analysis, viral particle titration. The heart of the Core is the Applied Biosystems 7500 and 7000 real-time PCR systems with software for primer selection and data analysis.
Real-time quantitative PCR provides improved sensitivity, accuracy, speed, and throughput over standard PCR methods. Detection is based on TaqMan fluorescent probes with 5' reporter and 3' quencher labels, such that synthesis from flanking primers leads to cleavage of the 5' reporter dye and detectable fluorescence. TaqMan probes can be synthesized with different color dyes permitting the analysis of multiple sequences in a single PCR reaction, so that each reaction can contain an internal control. The TaqMan probes can also be used for allelic discrimination assays. SYBR green can be used in place of the TaqMan probes to detect total PCR product.
To gain access to the facility and to book a time slot, follow these steps
- Go to http://researchcores.partners.org
- Create an account to access partners research cores (if you wish to use your partners password, do not enter any password and your partners password will be set as default)
- To add a fund #, select User, Fund, add new fund #, Assign Fund
- Select Cores, Real-Time PCR, MGH Quantitative Real-Time PCR, Schedule, Select Machine under equipment Menu and Schedule Time
- Once you have done this, contact our technician (email@example.com) and he or she can grant you access to the facility
Cristopher Bragg, Ph.D.
Location: room 6.025
This Core consists of a Zeiss LSM 5 Pascal laser confocal microscope with a Zeiss RGB vario laser module (458/488/514/543/633 nm) and Nikon C1 Confocal/TIRF System with 3 PMT and laser module (403/488/514/543 nm). A Zeiss Axiovert 200 fully motorized light microscope is available with fluorescence, bright-field, phase-contrast and Nomarski (DIC) capabilities. Image acquisition and analyses are performed using Zeiss LSM 5 Pascal Confocal Microscopy Software (Release 3.2) on 2 workstations. The Zeiss Physiology software is also available. Live cell imaging can be done using a Zeiss temperature controller with custom chamber and heating stage. The Nikon C1 Confocal/TIRF System fully motorized Nikon Eclipse Ti microscope is available with fluorescence, bright-field and TIRF capabilities. Imaging acquisition and analyses are performed using EZ-C1 and NIC-Elements Software. Live cell imaging is available using the temperature and CO2 controllers with custom chamber.
Examples of the specific services and analytical procedures available are:
• Double- and triple-labeling experiments
• Acquisition of high resolution images (2048 x 2048 pixels)
• Quantitative information for each channel
• 3-dimensional reconstruction
• Localized scans and FRAP (fluorescence recovery after photo-bleaching)
• Determination of intracellular ion concentration
• Visualization and analysis of intracellular transport mechanism
• Single molecule visualization, allowing dynamic observation and functional analyses of both in vivo and living cells
• Total internal reflection fluorescence experiments (TIRF)
• Dynamic events in live cells and tissues
Senior research technologist
Transmission Array Microscopy
Marian DiFiglia, Ph.D.
The transmission electron microscope (TEM) has been an essential tool for research in cell biology since its development in the 1950s and continues to aid in elucidating the complex architecture of the nervous system including membrane appositions between pre and postsynaptic structures, glia-neuron processes, glia-blood vessel contacts as well as the precise localization of membrane bound antigens by immunolabeling methods. The TEM is equipped with a digital camera that produces high contrast images. The facility is supported by other equipment associated with TEM use including ultra microtomes for thin sectioning, diamond knives, and a vibratome. Services include consultation, tissue processing, thin sectioning, and TEM analysis and image collection. Training for those interested in learning the techniques will be available.
Dr. Brian J. Bacskai
This is a light microscopic method of analysis that allows increased structural detail to be achieved at the light microscope level such as that associated with individual synapses. Preparation for array tomography requires the same tissue embedding equipment and ultramicratome used for TEM preparation. Tissue is embedded in a classical EM ultrastuctural media, cut into ribbons of 70 nanometer serial sections, then stained with multicolor immunofluorescence methods to distinguish subcellular epitopes. Images are acquired using a Zeiss Axio Imager Z2 microscope with coupled Photometrics CoolSNAP HQ2 CCD camera and a high resolution objective (63x 1.4NA Plan Apochromatic oil DIC). Images from the same position on each section of the ribbon are collected making a stack for 3-dimensional reconstruction of the subjects of interest. The Image analysis is performed in a semi-automated fashion using NIH software Image J, Matlab, and a series of simple programs written and made freely available by the inventor of the method. For information contact Steve Hou at Shou@partners.org.
in vivo Multiphoton Imaging
The technology of multiphoton microscopy allows imaging of visible light fluorophores within a living animal from the brain surface to a depth approximating layer III of mouse cortex. Scanning occurs in X, Y and Z directions for 3 dimensional rendering. Viral delivered fluorescent markers and small molecule fluorescent reporters are routinely used. The unit consists of a BioRad1024 multiphoton microscope with custom modifications mounted on an Olympus BX50WI upright microscope. A mode-locked Ti:Saphire laser (MaiTai) generates two-photon fluorescence with 800 nm excitation, and detectors containing three photomultiplier tubes (Hamamatsu Photonics) collect emitted light in the ranges of 380-480, 500-540, and 560-650 nm. The microscope stage has been modified to allow stereotactic frame placement, inhalant anesthesia, and temperature control. A surgical suite is located adjacent to the microscope facility and is equipped with all necessary tools for implantation of cranial windows for imaging.
For information and to get access to this facility, contact Dr. Steven S. Hou, Shou@partners.org.
For the past 40 years, continuous progress of microscale fabrication techniques has revolutionized the electronics industry. The same microscale technologies, now entering the field of biomedical sciences, are poised to bring the same revolutions to the 21st century biomedical sciences. A broad range of applications are targeted, ranging from point-of-care diagnostic devices to implantable micro-sensors, from tissue-engineered products to cell-based drug screening tools, and from gene chips to novel molecular biology tools. In this dynamic context, the Microfluidics Core provides know-how and state-of-the-art facilities for investigators in nervous system development, function, and dysfunction, to measure, control, and perturb the cellular microenvironment at the scale comparable to cell size.
Three key areas of expertise are supported by this Core
- microfluidic devices for cell motility studies,
- microfluidic devices for whole-cell and cell-organelle separation,
- microfluidic devices for axon guidance and high precision imaging studies.
The core will provide assistance to investigators with the design, fabrication and implementation of microfluidic tools for specific applications, and delve into entirely new, unexplored areas of applications.