The Unified Flow Core provides state-of-the-art flow cytometry and cell sorting services to the entire University of Pittsburgh research community as well as researchers at neighboring institutions. A skilled staff provides help with instrument setup, data analysis, and consultation for experiment design. Training is available to enable investigators and their staff to run the analytical cytometers themselves. The TGT (Transgenic and Gene Targeting) and Innovative Technologies Development (ITD) Cores are a shared resource designed to produce genetically engineered mice for investigators of University of Pittsburgh. Our laboratory provides a broad, comprehensive range of services to investigators. ITD Services For information about the ITD core services, or to initiate a CRISPR project contact: Dr. Sebastien Gingras, ITD Core Director, sgingras@pitt.edu. Genetic Engineering in mice using CRISPR/Cas9 technology: Consult with investigator on the design of the targeting strategy. Detailed analysis of the gene structure to provide the best targeting strategy to meet investigator’s objectives. We provide complete a turnkey service that includes project design, synthesis of sgRNA and Cas9 mRNA, assessment of reagents quality, editing efficiency and mouse genotyping. Classical transgenesis: Design of transgenic vectors. Assistance with gene editing in cell lines using CRISPR: We can provide design and assistance for genome engineering project in cell lines. We will advise the investigator on the best way to generate the desired cell lines. We will provide the design for a custom targeting strategy, identify targets sites, the design of targeting vectors, cloning procedure and genotyping strategy. Other/New: The Innovative Technologies Development Core will also consider developing new emerging technologies or other services that would be valuable to for investigators of University of Pittsburgh. TGT Services For information about the TGT core services, or to initiate a project contact: Dr. Chunming Bi, Core director, chunming@pitt.edu and Binfeng Lu, Faculty advisor, binfeng@pitt.edu CRISPR/Cas9 gene editing: We inject DNA/RNA produced by Innovative Technologies Development Core. Transgenic projects: In TGT lab, transgenic mice are generated by the microinjection of purified DNA. We can inject regular DNA and BAC DNA. ES cell injection: We can inject ES cells for investigators who have either generated targeted clones in their lab or obtained from another source. Currently we accept ready-to –be injected ES cells. Re-derivation: This process eliminates pathogens from infected mouse lines. Re-derivation allows the import of established mouse lines with either known pathogens or of unknown health status from anywhere in the world. Cryopreservation: Cryopreservation is the process of freezing germplasm for stable, long-term storage in liquid nitrogen. It is a safe method for preserving viable pre-implantation embryos or sperm that can be easily recovered at a future date to revive the line. Our cryo-recovery service allows the worldwide import and recovery of mouse lines from frozen sperm and embryos. Our ICSI system allows to recover mouse line from sperm with low motility. Long term storage of frozen germplasm: TGT lab accepts sperm and embryo which were frozen in other institutes, and store them in the liquid nitrogen for long term. IVF for CRISPR projects: Generate embryos by IVF for CRISPR/Cas9 gene targeting projects. A Bioplex II Luminex machine is available for use by labs affiliated with the Department of Immunology. This machine performs multiplex analysis of cytokines/chemokines as well as signal transduction pathways in a single small volume sample. Up to 41 cytokines/chemokines can be analyzed in a single sample. Individual labs wishing to use the instrument should contact Penelope Morel. At present, labs wishing to use the instrument will need to purchase the required bead kit and perform the assay themselves (the necessary equipment to perform the assays - magnet, vacuum manifold and shaker – is available in the Morel lab). Thus we have the capability to perform assays with magnetic and non-magetic beads. We will run the completed plates on the Luminex machine and provide the results to each investigator. Investigators who plan to use the machine frequently may be trained to run their own plates. There is no fee for this service but investigators who use the machine frequently will be asked to contribute to the purchase of the service contract needed to maintain this machine. For more information, please contact Penelope Morel. Scope and Mission: The Immunology Grant Pre-Review Committee provides constructive feedback on the Specific Aims section of NIH R01 and R21 submissions. We focus exclusively on R01s and R21s for highest impact. Outside of our current purview: critique of the Research Plan or submissions targeted to other funding mechanisms (NIH R03, non-profit, NSF, DOD, etc.). Both of these are future goals. Submission deadline: submit your Specific Aims at least 6 weeks prior to NIH due date. Turnaround time: The committee will provide you with critiques 7 business days after assignment to reviewers. Click here to download the Grant Pre-Review submission form. Contact Lisa Borghesi for further information Lisa Borghesi (412.383.7074). This is a CTL Immunopot elispot reader using an Image Acquisition version 4.5 and Immunospot version 5.0 analysis software. It is capable of performing basic and double-color counting. There are no fees to use this instrument. This system is used for digital imaging of western blots developed with ECL or fluoresent reagents in the red, green and blue spectrum. This equipment is available for departmental use only. No fees are applicable. For more information, please contact Larry Kane. Epoch™ has a monochromator-base UV-Vis wavelength selection. Wavelength range from 200 nm to 999 nm and 6- to 384- microplate reading capability, controlled with the Gen5 Data Analysis software interface. In addition, micro-volume quantification is fast and easy with the Take3 Micro-Volume plates. Pre-programmed nucleic acid and protein protocols in Gen5 make quantification fast and easy. For more information contact Pamela Beatty, 412-383-5287.Flow Cytometry
Transgenics and Gene Targeting
Luminex
Grant Pre-review
Elispot Reader
ProteinSimple FluorChem M Multifluor System
Epoch Microplate Spectrophotometer