VTMC Services and Facilities
CRISPR/Cas9-Directed Gene Editing
The VTMC has developed and is pleased to now offer CRISPR/Cas9-directed gene editing services utilizing next-generation dimeric RNA-guided FokI nuclease (RFN) technology. This system allows precise and efficient gene knockout via gene disruption and can be modified to introduce site-specific mutations into endogenous genes. While monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing, they can induce unwanted off-target mutations with high frequencies. Therefore we offer RFNs for which dimerization, rather than just co-localization, is required for efficient genome editing activity. Using this technology, the core has achieved a knockout efficiency of several different genes in cell lines ranging between 50% and 90%. The timeline from initial design to delivery of custom-targeting and associated expression plasmids for cell transfection is 2-3 weeks. The core also provides advice and services related to CRISPR technology, including design, screening, genotyping, and sequencing of modified cell clones.
Lentiviral Vectors for Use in Animals, Tissues and Cells
The VTMC provides well characterized third generation VSV-G pseudotyped lentivirus self-inactivating (SIN) vectors for use in cells, tissues, or intact animals. The lentivirus expression plasmid, pRRL-cPPT-CMV-X-PRE-SIN, incorporates a central polypurine tract (cPPT) and a posttranscriptional regulatory element from human hepatitis B virus (PRE). Expression plasmids encoding an enhanced GFP (eGFP) reporter, luciferase, or β-galactosidase with and without a nuclear localization signal serve as positive controls and quality controls. The Core has pioneered the use of IRES to provide coordinate expression of two or more genes of interest in bicistronic retrovirus vectors and has constructed three bicistronic vectors encoding encephalomyocarditis virus (EMCV) IRES, a multiple cloning site and a marker gene, enhanced GFP (pRRL-cPPT-CMV-X-EMCV-eGFP-PRE-sin). To achieve the goal of coordinate gene expression at equivalent and high levels the Core employs constructs encoding peptide 2A. These constructs enable affiliate investigators to monitor the expression of both their gene of interest and a marker gene. This is a major benefit where the investigational gene is not easily monitored and also permits determination of virus titer.
The Core also generates lentiviruses encoding promoters to achieve cell and tissue specific transgene expression for use in vitro and in vivo. For example, the Core has generated vectors encoding a hepatocyte specific promoter, and an adipocyte-specific adiponectin promoter. Furthermore, the Core generates lentiviruses encoding small hairpin RNA (shRNA) to provide sustained gene silencing in vitro and in vivo.
Viruses are purified and concentrated using a Tangential Flow Filtration system (Spectrum Laboratories) and are produced in serum free medium (OptiMEM or UltraCULTURE). Single filter/dialysis yields titers of 10^7 IU/ml volume of 35 ml from a 50 cell plate batch, and two filter/dialysis yield titers of 10^8 to 10^9 IU/ml volume of 1-2 ml from a 50 plate batch. All lentiviruses are titered and verified to lack replication competent virus before they are given to affiliate investigators.
Adenovirus Associated Vectors (AAV) for Use in Animals, Tissues and Cells
The VTMC provides well characterized, titered AAV vectors of serotypes AAV8/9 and AAVD/J. The latter serotype is particularly useful in providing high level transduction of heart, lung and brain tissue in vivo. Expression plasmids encoding marker genes such as enhanced GFP (eGFP) dsRed are available. The Core also generates AAV encoding small hairpin RNA (shRNA) to provide sustained gene silencing in vitro and in vivo.
We employ an AAV Helper-Free System (Cell Biolab) that allows the production of infectious recombinant human adeno-associated virus (rAAV) virions without the use of a helper virus. In the AAV Helper-Free System, most of the adenovirus gene products required for the production of infective AAV particles are supplied on the plasmid pHelper (i.e. E2A, E4, and VA RNA genes) that is co-transfected into cells with human AAV vector DNA. The remaining adenoviral gene product is supplied by the 293 host cells, which stably express the adenovirus E1 gene. By eliminating the requirement for live helper virus the AAV Helper-Free System provides a safer and more convenient gene delivery system. The AAV Helper-Free System can accommodate inserts of up to 3 kb. All AAV vectors are tittered and tested before they are given to affiliate investigators. AAV vectors are purified using a Transflow Filtration system (TFF) that generates virus titers of ~1013 IU per ml.
Recombinant adeno-associated viruses are important tools for gene delivery and expression. AAV has not been reported to cause any diseases. Together with its replication defective nature, AAV has a good safety profile to be used in gene transfer in vivo, and as potential gene therapy vehicles. Recombinant AAV is capable of infecting a broad range of cell types including non-dividing cells and remaining as concatemers for long-term expression. Compared with other viral vectors such as adenovirus, AAV elicits very mild immune response in animal models.
Replication Competent Retrovirus (RCR) Assay
The VTMC performs the RCR assay on all lentivirus preparations before they go out to affiliate investigators’ labs. The Core has not yet found replication competent virus in any lentivirus preparations, in agreement with reports in the literature.
Lentiviral Constructs for Generation of Transgenic Mice
Lentiviral vectors for generation of transgenic mice or mice expressing shRNA are generated by the tangential flow filtration system, purified and verified to be replication deficient. They will then be provided to the Transgenic Resources Program for generation of mice.
Retroviral Vectors for Use in Animals, Tissues and Cells
The VTMC generates and titers murine retrovirus vectors (Moloney Murine Leukemia Virus; MMLV), amphotropic virus or virus that infects human cells from PT67 or PG13 packaging cells and ecotropic virus from PE501 packaging cells for transduction of replicating cells. Helper-free amphotropic and ecotropic retrovirus is also produced by the Phoenix cell system. Expression plasmids encode the gene of interest and a selectable marker gene or reporter gene, or cell type-specific promoters, as described above for lentiviral vectors. Other packaging cells will be used if requested. For investigators requiring murine viruses of titers >10^6 IU/ml the virus is purified and concentrated through tangential flow filtration and produced in serum free medium (Optimem) from PT67 packaging cells to generate titers >10^8 IU/ml.
Adenoviral Vectors for Use in Animals, Tissues and Cells
The VTMC produces vectors for generation of recombinant adenoviral vectors via the DRC at the University of Chicago, in collaboration with Dr. Christopher Rhodes. Each user is consulted by the VTMC core directors and Dr. Rhodes as to the options available for the recombinant adenovirus they intend to generate. The VTMC has access to a variety of adenoviral shuttle vectors that are engineered to express genes in a generic, cell specific, biscistronic, or inducible (tet on/off), as well as rhRNAs. The VTMC inserts the gene of choice or shRNA into one of these shuttle vectors and verifies that it is in frame and appropriate by DNA sequencing and preliminary expression studies. That shuttle vector plasmid is then sent to the Rhodes’ laboratory, where the recombination with the adenoviral genomic vector (type V E1A and/or E3 deficient), plasmid preparations, recombinant adenoviral generation, amplification and purification are done. The final recombinant adenovirus preparations are of high quality and titer (~10^13 pfu/ml). Two thirds of the final purified (ready to use) adenoviral preparation is provided to the UW DRC affiliate investigator. Each recombinant adenovirus provided is plaque-purified and are of the highest quality and titer possible.
Constructs for Generation of Genetically Engineered Mice Using Non-Viral Methods
The VTMC provides non-viral vectors for generation of transgenic, knockout, and knockin mice. The standard targeting vector for generation of knockout mice is based on the pBluescript II KS (-) construct (Fermentas), which allows for tissue-specific Cre-Lox-mediated deletion. The Core director meets with the affiliate investigator to choose a critical exon in the gene of interest to be flanked by loxP sites. The purified vector is delivered to the Transgenic Resources Program. Non-conditional knockout plasmids are designed to delete the entire coding sequence of the target gene. The VTMC carries out DNA sequencing, through the UW Biochemistry Sequencing Core, to verify fidelity of all vectors before sending them to the Transgenic Resources Program or affiliates.
Specialized Molecular Biology Methods Not Routinely Performed in Affiliates Laboratories and Limited Genotyping of Generated Mice
The Core offers real-time PCR with mRNA copy number quantification (using a Stratagene Mx4000 quantitative PCR instrument), cloning and targeting vector design and construction, site-directed mutagenesis, and construction of plasmids for virus and mouse production. The Core offers gene synthesis either in-house or using the local company Blue Heron Biotechnology. This option can be especially useful in accurately obtaining long genes, vectors, or complex constructs, when no native sequences are available or when rare RNA transcripts are required.
Cost-effective Production of Transgenic and Knockout Mice at the UW Transgenic Resources Program
DRC affiliate investigators using the VTMC are able to use the Transgenic Resources Program at the UW at a competitive price to effectively generate transgenic and knockout mice. The VTMC offsets the cost for DRC affiliate investigators to use the Transgenic Resources Program.
An important aspect of the VTMC’s function is to offer consultation concerning virus production and use, specific viruses that best satisfy each affiliate investigator’s needs, generation of genetically engineered mice, molecular biology techniques, e.g. qRT-PCR with primer/probe design, and sources to obtain constructs and ES cells for transgenic and gene targeted mice. These services are provided by the core directors. Consultation in using repositories and other resources available for faster generation of mice is also provided. In addition, the VTMC offers training in any technique that is carried out by the VTMC.
The VTMC’s new laboratory (710 sq. ft) is on the first floor of the University of Washington at South Lake Union. The VTMC space consists of wet lab space, tissue culture room, two procedure rooms, and data processing rooms.
Key equipment within the VTMC includes PCR machines (Stratagene), a Nanodrop, and an Mx4000 system. The VTMC has access to a Tangential Flow Filtration system for virus purification through the Department of Neurological Surgery as well as access to shared equipment in the Diabetes and Obesity Center of Excellence, including beta-counters, gamma-counters, ABI real-time PCR machines, MaxWell RNA/DNA isolators, fluorescence and luminescence plate readers, gel scanners, and flow cytometers and cell sorters.