The Vector Development and Production Core provides viral vectors with custom-designed promoters and reporter genes and capacity for gene regulation.
Provides viral DNA packaging and purification services.
Reporter genes include: Enhanced green fluorescent protein (eGFP), red fluorescent protein (dsRFP), luciferase, alkaline phosphatase or lacZ and a multiple cloning site (MCS) downstream of a cytomegalovirus (CMV) IE promoter.
The Core maintains high quality stocks of packaging plasmids and carries out packaging and “purification”, titering and allocation of vectors for the investigators.
The Core will provide appropriate lentivirus, retrovirus and AAV plasmid backbones, construction information and assistance to investigators, as well as packaging vectors for them.
Investigators using the lentivirus vector will be supplied with a plasmid backbone carrying multiple cloning sites (MCS) downstream of a cytomegalovirus (CMV) immediate early (IE) promoter. Other plasmids are also available with a reporter gene such as GFP, RFP under an IRES element. These vectors will express both the transgene of interest as well as the reporter gene.
For investigators interested in retrovirus vectors, a number of constructs are provided, including drug selection cassettes for puromycin, zeomycin and neomycin, as well as reporter genes, eGFP or luciferase, and cloning sites for transgenes of interest under the 5' LTR promoter.
AAV vectors made available through the core will carry AAV2 ITR elements serotyped with either AAV1, 2, 5, 8, 9, or rh10 capsids. A number of tissue-specific promoters are available together with the constitutively active CBA promoter. A bicistronic vector expressing the transgene as well as GFP is available to facilitate identification of vector-transduced cells in cell culture and in vivo. Basic vectors encoding EGFP, firefly luciferase (Fluc), Gaussia luciferase (Gluc), and Cre recombinase (under CBA or Synapsin-1 promoters) are available.
Investigators will be responsible for inserting their genes of interest into the MCS, validating correct construction by restriction analysis, testing bioactivity by transfection assays (when possible), and supplying clean maxi-prepped DNA for packaging.
The Core will maintain high quality stocks of packaging plasmids and will carry out packaging and “purification”, titering and allocation of vectors for the investigators.
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.