Lentiviral Vector Services

Lentiviruses (LV) are remarkably complex relative to the other retroviruses, encoding an additional six viral proteins: tat, rev, vpr, vpu, nef, and vif. Lentiviruses do not generate any potent cellular immune response, making them an excellent tool for in vivo gene delivery. A disabled version of human immunodeficiency virus (HIV) has been developed as a lentivirus-like vector for this purpose.

LVs infect non-cycling cells. Lentiviral particles can often be pseudotyped with the envelope of the Vesicular stomatitis virus (VSV), allowing lentiviral vectors to introduce genes into a broad range of tissues. Lentiviral vector-mediated transgene expression can last for several months without detectable pathology.

Service Packages:

1. Production of recombinant LV particles and delivery of purified, ready-to-use viral supernatants at titer of 10⁶-10⁷ TU/ml.

2. Construction and delivery of custom lentiviral vector carrying desired gene or siRNA with GFP or RPF reporter.

Finished lentiviral vector constructs	Viral vectors
High titer, ready-to-use viral supernatants	Detailed viral vector construction maps

3. Construction of custom lentiviral vectors carrying the desired gene or siRNA with GFP or RPF reporter and transfection of customer-supplied cell line(s).

Finished lentiviral vector constructs	Viral vectors
High titer, ready-to-use viral supernatants	Detailed viral vector construction maps
Three vials of stable cells, 5×10⁶ cells in total	Detailed protocols and procedures

Services (SC1168):

1. Recombinant lentiviral vector construction: GenScript uses a three-part assembly system to generate recombinant lentiviral vectors, promoters, core vectors for packaging, and transgenes with and without reporters. We currently offer two standard options:

GenScript can use a stock promotor, CMV, C-Fos, PGK, EF1a and MPSV, or Ubiquitin, with the transgene cloned upstream of the IRES-GFP in our shuttle vector pENTR 5' TOPO TA (Invitrogen Corp.). The concurrent expression of the GFP reporter provides a convenient monitoring means of host cell infection.
GenScript can use custom promoter with a stock reporter gene, GFP or luciferase, already cloned into a blank plasmid pENTR 2B (Invitrogen Corp.)

Note: it is customer's responsibility to provide the transgene or promoter within the correct plasmids. If the transgene is not physically available, GenScript can synthesize it or clone it from rodent or mouse cDNA libraries.

2. Recombinant LV production: Recombinant LV preparation is similar to the production of simple retroviral vector systems. We currently use three-plasmid transient co-transfection of 293T cells to produce recombinant viral vectors: the helper-packaging construct (HIV gag-pol, rev, and tat), the envelope plasmid (env), and a lentiviral transfer vector. The transfected cells are incubated for 72 hours to allow LV particle production in the cell supernatants. The supernatants are then filtered and pelleted by sucrose gradient, and virus titer is estimated by FACS analysis on infected cells (for those expressing GFP) or by Southern blot.

3. Recombinant lentiviral vector characteristics: Routinely, a single rLV stock is prepared starting from 30×150 mm-cell culture dishes.

Yield: 1×10³ viral particles/cell
Biological titer:10⁶-10⁷ TU/ml (concentrated)
Final volume: 1 ml of concentrated recombinant viruses in phosphate buffered saline (PBS)
Storage: The recombinant LV vector batches are stored at -80°C

4. Recombinant lentiviral QC assays: The infectious titer of recombinant lentiviral vectors containing GFP or LacZ is analyzed by fluorescence microscope or X-gal staining of transduced 293T cells. Alternatively, p24 can be examined by ELISA. Some of the viral stocks can also be tested for the presence of replication-competent lentivirus by monitoring p24 expression for 30 days in the culture medium of transduced 293T cells. Upon request, GenScript can provide service of replication competent virus assay.

Additional Services:

GenScript offers ready-to-use LV-GFP and LV-LacZ reporter vectors that can be used as controls.

Delivery Specification and Timeline

Quotations and Ordering:

For quotation requests, please download and complete GenScript Viral Vector Services Quotation Request Form, and email or fax it to us with a detailed description of project requirements. For questions, please use our secure web server. However, you may also contact us by email at order@genscript.com, phone, or fax.

Orders can be placed by phone, email, or fax with either a formal PO (Purchase Order) or credit card. Please email or fax us a completed GenScript Viral Vector Services Quotation Request Form. Our customer service representatives are available 24 hours Monday through Friday. If target gene is physically available, please submit your target gene along with a hard copy of completed GenScript Viral Vector Services Quotation Request Form to Viral Vector Services, GenScript Corp., 120 Centennial Ave., Suite 105, Piscataway, NJ 08854, US.

	Order by email: order@genscript.com
	Order by phone: 1-877-436-7274 (Toll-Free) 1-732-885-9188
	Order by fax: 1-732-210-0262 1-732-885-5878