Washington CRISPR, Vector and Transgenic Mouse Core

Overview

The overall goal of the Vector and Transgenic Mouse Core is to provide Diabetes Research Center affiliate investigators with state-of-the-art vectors necessary to overexpress, knockdown, knockout, or alter expression of RNAs and proteins of interest in cultured cells, isolated tissues, and animals.

To accomplish this goal the Vector and Transgenic Mouse Core provides the following to DRC affiliate investigators:

• Production of lentivirus vectors, adeno associated virus (AAV) vectors, retrovirus (MMLV) vectors and adenovirus vectors for use in animals, tissues and cells;
• CRISPR/Cas9-directed gene editing;
• Production of lentiviral constructs for generation of transgenic and knockdown mice;
• Production of non-viral vectors for generation of transgenic, knockout, and knockin mice;
• Specialized molecular biology methods not routinely performed in affiliate investigators’ laboratories;
• Cost-effective production of genetically engineered mice through the University of Washington (UW) Transgenic Resources Program;
• Consultation and training

Services

CRISPR/Cas9-Directed Gene Editing

The Vector and Transgenic Mouse Core has developed and is pleased to offer CRISPR/Cas9-directed gene editing services utilizing next-generation dimeric RNA-guided FokI nuclease (RFN) and Cas9 nickase technologies. The RFN system allows precise and efficient gene knockout via gene disruption. 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.

Adenovirus Associated Vectors (AAV) for Use in Animals, Tissues and Cells

The Vector and Transgenic Mouse Core provides well characterized unique packaging plasmids for 12 different capsid serotypes including AAV serotypes 1-9, and hybrid serotypes DJ, DJ8/9. The DJ serotypes are particularly useful in providing high level transduction of heart, liver, lung and brain tissue in vivo. Expression plasmids encoding marker genes such as enhanced GFP (eGFP) and dsRed are available. Cell-type selective promoters are available for tissue-selective expression in vivo. 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 4 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 ~1012 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.

Lentiviral Vectors for Use in Animals, Tissues and Cells

The Vector and Transgenic Mouse Core 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.

Replication Competent Retrovirus (RCR) Assay

The Vector and Transgenic Mouse Core 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.

Retroviral Vectors

The Vector and Transgenic Mouse Core 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 >10^7 IU/ml. Retroviral vectors are often used for replicating cultured cells, but can also be used in certain in vivo models if replicating stem cells are targeted.

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

Diabetes Research Center affiliate investigators using the Vector and Transgenic Mouse Core are able to use the Preclinical Research and Transgenic Services Program at the UW at a competitive price to effectively generate transgenic and knockout mice. The Vector and Transgenic Mouse Core offsets the cost for Diabetes Research Center affiliate investigators to use the Preclinical Research and Transgenic Services Program.

Consultation

An important aspect of the Vector and Transgenic Mouse Core’s function is to offer consultation concerning virus production and use, specific viruses that best satisfy each affiliate investigator’s needs, CRISPR/Cas9-directed gene editing, 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 Vector and Transgenic Mouse Core offers training in any technique that is carried out by the Vector and Transgenic Mouse Core.

Facilities

Laboratory

The Vector and Transgenic Mouse Core’s laboratory (710 sq. ft) is located on the second floor of the University of Washington at South Lake Union. The Vector and Transgenic Mouse Core space consists of wet lab space, tissue culture room, and procedure room.

Equipment

Key equipment within the Vector and Transgenic Mouse Core includes PCR machines (ABI), a Nanodrop (Thermo), Mx3005P and Mx4000 real-time PCR systems (Aglilent), and a Tangential Flow Filtration system for virus purification. The Vector and Transgenic Mouse Core has 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.