Recombinant Viruses: Production, Purification, Concentration, Validation and Titer
Starting from an appropriate donor plasmid, the GTC can produce recombinant lentiviruses, retroviruses, and adeno-associated viruses.
Retroviruses & Lentiviruses
A typical preparation is from a 10 cm dish of HEK293 cells, but multiples of this size preparation are encouraged. Typical yields from one 10 cm dish range from 107to 108IFUs. The recombinant viruses are packaged with the VSV-g coat protein. They are concentrated by PEG precipitation and resuspended in less than 300 ml of PBS. The viruses are titered by quantitative PCR of virus-specific sequences from genomic DNA prepared from virus-infected cells. This ensures that actual IFUs are assayed, not just empty or damaged virions or free DNA as some assays report.
Both 2ndand 3rdgeneration lentiviruses can be produced. If requested, the virus preparation can be assayed for replication deficiency.
Retrovirus/Lentivirus production and concentration
$100 per 10-cm plate
$100 per titer
Assay for viral replication deficiency
$100 per viral prep
A typical preparation is from ten T225 flasks and generates 1012to over 1013packaged genomes. A variant of serotypes can be produced, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. The recombinant virus is purified on an iodixanol step-gradient, washed in PBS and suspended in less than 500 ml of PBS. Titering is done by quantitative PCR of virus-specific sequences.
Adeno-associated virus production, purification, and concentration
$1500 per preparation
Construction of Recombinant Plasmids
Recombinant plasmids to meet the experimental needs of the investigator can be constructed. Consult with the Core Director on the construction strategy. Many different promoters and tags are available.
Recombinant plasmid constructions
$500 per plasmid (+ cost of special materials)
Stable Cell Line Construction
Recombinant constructs designed by the investigator can be integrated into mammalian cell lines to produce new cell lines with novel properties. The GTC uses both lentivirus and piggyBac transposons for stable cell line production.
Stable cell line construction
$100 per cell line (once suitable recDNA has been obtained)
(to be determined after consultation with GTC director.)
The GTC can purify plasmid DNA from existing bacterial strains or from plasmid DNA stocks. Average yield is over 500 mg (yield is dependent on plasmid copy number).
Plasmid DNA maxiprep
$50 per plasmid (from bacterial stocks)
$55 per plasmid (from plasmid DNA stock)
$6.00 per sequencing run
Oligonucleotidedesign and synthesis
$10 per oligonucleotide
*Some projects may require the purchase of specialized materials or outside services. The costs of these materials will be billed to the investigator.
**Projects that require special materials will incur an additional cost for the purchase of such materials.