Lentiviruses have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a huge advancement in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo. As the particles are often pseudotyped with the envelope of the vesicular stomatitis virus (VSV), the vector can serve to introduce genes into a broad range of tissues and can be used in vivo. Furthermore, it has been demonstrated that in vivo expression is sustained for several months without detectable pathology. In the original lentivirus vectors described by Naldini et al (Naldini et al., 1996a) (Naldini et al., 1996b) , the vector pCMVΔR8.2 supplies all but the HIV envelope in trans, the vector pMD.G is used to produce the VSV-G pseudotype, while the transgene (lac Z) is inserted into the plasmid pHR’, which uses a lentiviral LTR, a splice donor and gag splice donor and acceptor associated with a rev responsive element. These three plasmids are used to transduce 293T cells and the supernatant assayed for p24 gag activity.
From these original experiments, new plasmids have been devised which have more versatile features. These include such plasmids as pLenti4/V5-DEST™, pLenti6/V5-DEST™, pLenti6.2/UbC/V5-DEST™ (Invitrogen Life Technologies Inc.) which utilize Gateway Technology™ (Invitrogen Life Technologies Inc.) in which recombination occurs through the attR1 and attR2 and thus allows for rapid cloning Lentiviral systems for gene delivery are generally derived from the human immunodeficiency virus. The VDL utilizes a vector construct that employs a nonhomologous viral envelope (derived from the vesicular stomatitis virus), possess only three HIV genes, and separates the genes encoding the components necessary for viral packaging onto four different plasmids. These features, in conjunction with several other biosafety enhancements, prevent recombination of replication-competent HIV.
All lentivirus vectors produced in the VDL are produced in compliance with GLP standards. The production of a lentivirus virus begins with cloning the gene of interest (goi) into an entry plasmid of the Gateway Technology™ (Invitrogen). The entry plasmid containing your gene of interest is then recombined with one of the ViraPower™ Lentiviral Expression plasmids using Gateway Technology™ (Invitrogen). The ViraPower plasmids which lacks the gag/pol envelope genes and other accessory genes. The expression plasmid is cotransfected into 293FT cells with three supercoiled packaging plasmids (pLP1(gag/pol), pLP2 (rev) and pLP/VSV-G (VSV-G envelope) which supply helper functions and viral proteins in trans. At 48-72 hours post-transfection, the supernant (containing the viral particles) is harvested and clarified. At this point lentiviral vectors can be used to transduce the mammalian cell line of choice.
Read more about the Virapower™ Lentiviral Expression System.
Quality control/assurance testing for lentivirus vectors includes, titering and test for sterility and test for endotoxin.
In addition to providing the above mentioned services, the VDL also offers Pseudotyped Lentiviral Vectors.
- Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996a). Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A. 93(21), 11382-8.
- Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996b). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259), 263-7.