Trobridge Lab

Lentiviral Vector Production

Description:

We can assist WSU investigators with lentiviral vector production and titering. We produce replication-incompetent vector preparations by transient transfection using vector plasmid DNA and second generation packaging helper plasmids. These vectors can then be used for stable expression of transgenes in target cells. This approach is particularly useful for difficult to transfect cell types including primary cells. We typically produce vector preparations in the range of 107 to 108 transducing units/ml (TU/ml) for the vectors shown below. For vector plasmids containing a fluorophore for detection we use flow cytometry to determine viral titer in transduced HT1080 cells (functional assay). If a fluorophore is not present in the vector plasmid we use an ELISA to determine viral titer indirectly using a viral protein, p24 in vector preparations.

The production process takes two weeks including determination of vector titer by functional assay. A Lentiviral vector production request form must be turned in no later than 4pm Thursday the week before you would like vector production to begin.


First-time users (PI):

We will schedule a meeting with the Director to discuss lentiviral vector production and usage. We request the PI be present for this first meeting. Small aliquots of fluorescent vectors are available to new users (PIs) to evaluate gene transfer and expression in your cell type of choice. http://www.pharmacy.wsu.edu/labs/trobridge/lvp.html

Overview of lentiviral vectors:

Lentiviral vectors, derived from the lentiviruses subclass of the family Retroviridae, are a common tool used to deliver genetic material into cells. We use lentiviral vectors derived from HIV-1. HIV-1-derived lentiviral vectors have a large packaging capacity and can transduce both dividing and non-dividing cells.

In order to produce safe, replication-incompetent lentiviral vectors, helper plasmids have had accessory genes of HIV-1 not required for vector production removed. In order to further reduce the risk that recombination of helper plasmids could produce a replication competent provirus, lentiviruses are rendered self-inactivating due to a deletion in the U3 region of the 3' LTR necessary for transcription. Upon reverse transcription and integration in transfected cells this deletion is transferred to the 5' LTR. This results in loss of viral transcriptional activation in the provirus LTR, allowing use of an internal promoter of choice. These vectors are pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) which allows efficient transduction of essentially all mammalian cells.
vector_production
  Vector production
SFFV-mCherry CD34 transduced (112K)
  Human CD34+ hematopoietic cells transduced with pRSC-SChW vector.
CMV-EGFP transduced no border (103K)
Human HT1080 fibrosarcoma cells transduced with pLenti CMV GFP blast vector.


Links to lentiviral vector resources

Maps of standard vectors:


Addgene#: 12252 - pRRLSIN.cPPT.PGK-GFP.WPRE

PGK-EGFP (9K)
pRRLSIN.cPPT.PGK-GFP.WPRE vector: Contains a phosphoglycerate kinase (PGK) promoter driving expression of enhanced green fluorescent protein (EGFP), as well as a safety modified woodchuck hepatitis post-transcriptional regulatory element (WPRE,W).

Addgene#: 17445 - pLenti CMV GFP Blast

CMV-EGFP-BLAST (16K)
pLenti CMV GFP Blast vector: Contains a cytomegalovirus (CMV) promoter driving expression of enhanced green fluorescent protein (EGFP), as well as a safety modified woodchuck hepatitis post-transcriptional regulatory element (WPRE,W). This vector also contains a Simian vacuolating virus 40 (SV40) early promoter and the prokaryotic EM7 (EM7) promoter driving expression of a blasticidin (Blast) resistance gene.

Trobridge Lab - pRSC-SChW

SFFV-mCherry (10K)
pRSC-SChW vector:Contains a spleen focus-forming virus (SFFV) promoter driving expression of a red fluorescent protein, mCherry (mCh). This vector also contains the safety modified woodchuck hepatitis post-transcriptional regulatory element (WPRE,W).


Forms:
Lentiviral vector production FAQ
Lentiviral vector production request form

Contact the Director:

  Grant D. Trobridge Ph.D.
 grant.trobridge@wsu.edu