Advances in techniques for efficient cellular engineering using virus-based gene delivery vehicles ("vectors"), have made it feasible to utilize gene transfer as a methodology for elucidating functions of specific genes by examining consequences of their over-expression or inhibition. The objective of the UCLA Vector Core is to promote and facilitate basic and translational research by providing investigators with access to vector technologies that enable efficient gene transfer to mammalian cells in culture and in in vivo.
We offer various pre-made lentiviral, vector stocks expressing standard marker genes to utilize in preliminary experiments. We construct and produce custom viral vectors that contain a specific sequence of interest (including wild type and mutant cDNAs with or without epitope tags, dominant-negative expression constructs, antisense mRNAs, siRNAs, etc.) for individual researchers.
Contact information (email preferred)
Emmanuelle Faure-Kumar, PhD, [email protected]
Janet Treger, PhD, [email protected]
Description of Services
- Project consultation: The first consultation prior to initiation of each project is to provide the investigator/staff with information about the Vector Core services, prices, types of vectors available, estimated timeline for vector production. Subsequent consultations are also available and indicated, especially in circumstances when there is a change or addition of services to the initial proposed project.
- IBC protocol: We assist PI/ staff with templates and pre-review for submission of research protocols to the UCLA Institutional Biosafety Committee (IBC).
- Support documents: Letters for grant and fellowship submissions, preliminary data showing feasibility of gene transfer with the vectors already available in the Vector Core and technical protocols, are available to investigators who have already obtained an initial consultation.
Small scale lentivector and adeno-associated virus (AAV) vector
- Non-concentrated adeno-associated virus (AAV): These preps are produced as small-scale after transient transfections of 293T cells. We offer the packaging plasmids for AAV-2, 2/8 and 2/9. For any other serotypes, the PI will have to provide the packaging plasmids. From a 10 cm dish, we collect 500ul of virus at concentration 5.10e10 to 5.10e11 VG/ml. The titration is done by qPCR using Takara kit.
Large scale lentivector
- Concentrated lenti/retro vector marking stocks: A collection of pre-made cryopreserved lentiviral and retroviral vectors expressing marking genes (GFP, DsRed, Luciferase) or selectable genes (TK, neoR) produced after transient transfection of 293T cells are available. High concentration lentiviral vectors typically range in concentration between 5 - 20 mcg p24/ml, available as 1 ml aliquots.
- Concentrated customized lentivector production: Lentiviral vectors expressing transgenes that are not extensively used or in conditions that are not routine (e.g. stored in “low serum” conditions) have to be requested individually. The concentration of virus is performed by ultracentrifugation of a large-scale (320 ml) production. Prior to large-scale production, vectors not previously tested by the Vector Core have to be produced at least once by a small-scale run.
- Plasmid DNA for vector construction: The Vector Core can clone into plasmid DNA corresponding to transfer vectors or containing transgenes or regulatory elements of interest (promoters, enhancers, IRES, etc) the gene of interest brought by the PI.
- Bacterial transformation for DNA amplification: If the PI/staff provide the plasmid DNA to be used in subsequent sub-cloning procedures, bacterial transformation can be performed for plasmid amplification.
- Maxi-prep and restriction enzyme analyses: In order to assure quality control, maxi-preps for subsequent sub-cloning procedures or virus production are preferentially produced in the Vector Core.
- Direct sub-cloning and analytical confirmation: In many cases, custom vectors can be constructed by direct sub-cloning. This requires the following steps: 1) restriction digest of insert DNA and vector, 2) gel-purify fragments, 3) dephosphorylate vector backbone, 4) ligate insert + vector fragments, 5) bacterial transformation and clone selection, and 6) mini-prep and screen for positive clones by restriction digest. Whenever possible, an electronic sequence and map of the expected final vector will be generated.
- PCR sub-cloning and analytical confirmation: In some cases, the DNA to be used as the insert can only be obtained by PCR. This requires the following steps: 1) design and purchase primers and perform PCR amplification of insert sequence (with Pfu polymerase to avoid mutations), 2) gel-purify PCR product, 3) restriction digest and dephosphorylate vector backbone, 4) ligate insert + vector fragments, 5) bacterial transformation and clone selection, and 6) mini-prep and screen for positive clones by restriction digest. Whenever possible, an electronic sequence & map of the expected final vector will be generated.
- Sequencing (300-600bp) of insert in lentivirus: We generally advise users to confirm the sequence of inserts sub-cloned into transfer vectors, or at least the ligation joints in the case of very long inserts. We will design the primers, send the mini-preps to sequencing and confirm the accuracy of the sequence. Primer production and sequencing are outsourced and charged back to the investigator.
Target cell transduction and expression analysis
- Transduction in six well plate of standard cell line (293T): Transduction of the vector to be tested is performed in six well plates in parallel to controls (mock control and a positive virus control expressing a marker gene). For lentiviral vectors, we recommend that gene expression analyses be performed three or more days after transduction to avoid artifacts such as pseudo-transduction.
- Transduction in six well plate of cell line provided by user: The PI/staff can also provide the virus with specific cell lines and/or primary cells along with the culture medium and growth factors required. Transduction of the vector to be tested is performed in six well plates in parallel to controls (mock control and a positive virus control expressing a marker gene). For lentiviral vectors, we recommend that gene expression analyses be performed at least three to five days after transduction to avoid artifacts such as pseudo-transduction.