Genetic Technologies Services

Genetic Technologies Services

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 10to 10IFUs. The recombinant viruses are packaged with the VSV-g coat protein.  They are concentrated by PEG precipitation and resuspended in less than 300 μl 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 2nd and 3rd generation lentiviruses can be produced.  If requested, the virus preparation can be assayed for replication deficiency.  

 

Retrovirus/Lentivirus production and concentration

$85 per 55-cm2 plate

Retrovirus/Lentivirus set-up

$25

Viral Titering

$110 per titer

Assay for viral replication deficiency

$110 per viral prep

 

Adeno-associated Viruses

A typical preparation is from ten T225 flasks and generates 1012 to over 1013 packaged 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 μl of PBS.  Titering is done by quantitative PCR of virus-specific sequences.

 

Adeno-associated virus production, purification, and concentration

$1650 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

per quote

 

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

$110 per cell line (once suitable recDNA has been obtained)

 

Site-Directed Mutagenesis

Investigator-designed amino acid changes, deletions or other changes to existing constructs can be constructed.  Consult with the GTC director regarding the feasibility and cost of the project.

 

Site-directed mutagenesis

(to be determined after consultation with GTC director)

 

Plasmid purification

The GTC can purify plasmid DNA from existing bacterial strains or from plasmid DNA stocks. The average yield is over 500 μg (yield is dependent on plasmid copy number).

Option 1  A plate containing bacterial colonies expressing the plasmid of interest is supplied along with ~200 ml of sterile media containing the appropriate antibiotic, a 50 ml sterile flask, and a 500 ml sterile flask.  The plasmid of interest is produced with these reagents. 

Cost is $90.00 per plasmid prep.

 

Option 2  A flask containing ~200 ml of freshly grown cells expressing the plasmid of interest with the appropriate antibiotic is supplied.  The culture should have an OD260 of 2.0 to 2.4.  The culture must be supplied prior to noon of the day the plasmid prep is to be made.

Cost is $65.00 per plasmid prep.

The average yield is over 500 μg of plasmid DNA, however the yield can vary dependent on the plasmid copy number. 

 

Miscellaneous 

DNA sequencing

Cost of material plus 20% handling surcharge

Oligonucleotide design and synthesis

Cost of material plus 20% handling surcharge

 

*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.

 

No