NGEC Cell and Virus Core Facility
The Northwest Genome Engineering Consortium Cell and Virus Core centralizes the specialized knowledge and equipment required for cell separation and analysis and NIL vector production activities.
Functions
The Cell and Virus Core serves three distinct functions within the NGEC. First, it assists investigators with cell analysis and separation. These activities include separation of surface expressed LAGLIDADG homing endonuclease variants generated by Scharenberg Lab experiments, isolation of hematopoietic stem cells from murine and canine sources and assisting with analytical flow cytometry to support various experiments in NGEC labs.
Second, the Cell and Virus Core assists with the construction and production of NIL vectors. As part of this function, the core provides: 1) consultation regarding the performance of various vector/donor/promoter combinations for individual experiments, 2) provision of established gene repair NIL vector backbone plasmids, 3) direct support for design and cloning of more complex NIL vectors, 4) small- and large-scale production of NIL vectors, 5) quality-control analysis of the NIL vectors including FACS- and ELISA-based titering and other relevant aspects of viral storage and delivery to NGEC laboratories.
Finally, the core is working to develop improved vector production and purification methods and improved vector systems. As lentiviral production methods advance and improved vector production systems become available, core personnel will incorporate these technologies into production protocols. In addition, core personnel will develop knock-in vector systems for murine XID and canine 494F18 and PK integration sites.
Instrumentation
The Cell and Virus Core houses a new BD FACS Aria flow cytometer for performance of all flow-cytometry analyses related to NGEC activities. This instrument is equipped with five lasers (355 nm, 405 nm, 488 nm, 561 nm and 640 nm) and appropriate detectors for 15-color/17-parameter analyses. It also is capable of two- or four-way bulk sorting into a variety of tube sizes, and has an aerosol management option to control the risk of biohazard exposure. The 355 nm laser allows for isolation of bone marrow “side populations,” which are highly enriched in long-term, repopulating hematopoietic stem cells, and the 561 nm laser allows enhanced detection of phycoerthyrin and related fluorophores, which increases the separation capabilities of the instrument for use in LHE sorting.
The core also supports an Applied Biosystems (ABI) 3730 DNA Analyzer for all NGEC sequencing needs. This instrument has 48 capillaries and produces up to 1,200 bases for each sequence, depending on the selected run length. Required sample volume is 10-50 microliters, housed in 96 well plates. Up to 16 plates can be housed in the Integrated Plate Stacker for automatic processing.
The Cell and Virus Core also has a dedicated Optima L-90K preparative ultracentrifuge, a Spectrum Labs KrosFlow Research II MiniKros Sampler-plus tangential flow filtration system and four tissue culture hoods dedicated for use in lentiviral production.
The NGEC Cell and Virus Core is located alongside the Flow Cytometry Core Facility at Seattle Children's Research Institute. Instrumentation in the Flow Cytometry Core Facility includes a first-generation BD FACS Aria flow sorter, a five-laser BD LSRII digital analytical flow cytometer (that can be used in parallel with the five-laser Aria to identify sort populations) and a BD FACsCalibur flow cytometer.