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Lentivirus

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.

Lentiviral vectors can now be used for a wide variety of in vivo and in vitro for high efficiency and permanent gene transfer.  Additionally lentiviruses provide a great deal of versatility for use with overexpression, siRNA, cre-lox and tetracycline inducible function, and cell-type specific promoter uses.

References

  1. 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 A93(21), 11382-8.
  2. 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. Science272(5259), 263-7.
  3. Singer O, Tiscornia G, Ikawa M, Verma IM. Rapid generation of knockdown transgenic mice by silencing lentiviral vectors. Nat Protoc. 2006;1(1):286-92.
  4. Tiscornia G, Singer O, Verma IM. Production and purification of lentiviral vectors. Nat Protoc. 2006;1(1):241-5.
  5. Tiscornia G, Singer O, Verma IM. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc. 2006;1(1):234-40.
  6. Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc Neurosci. 2006 Nov;Chapter 4:Unit 4.21. Review.
  7. Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc Hum Genet. 2007 Jul;Chapter 12:Unit 12.10.

Adeno-Associated Virus (rAAV)

Recombinant adeno-associated virus (rAAV) is a widely utilized vector for gene delivery into mammalian cells. rAAV is ideal because of their ability to infect both dividing and non-dividing cells, and establish long-term transgene expression in a large of variety of tissues and cells. They are nonpathogenic and nonimmunogenic and have a wide variety of serotypes. rAAV has a high transduction efficiency both in vivo and in vitro.

The Viral Vector Core currently provides rAAV serotypes 1, 2 and 5. We use either a 2-plasmid system or a 3-plasmid system for transfection in 293T cells, depending on serotype of rAAV that you order. rAAV is harvested and purified by density gradient ultracentrifugation using an iododexanol gradient, and then further purified and concentrated by dialysis.  rAAV titer is confirmed with quantitative PCR (qPCR).  Your AAV order will be divided into small individual-use aliquots and stored in phosphate buffered saline (PBS) at -80 until ready for pickup. Please call or email us for any questions concerning AAV production.

References

  1. Ayuso, E., F. Mingozzi, et al. (2010). "High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency." Gene Ther 17(4): 503-510.
  2. Grimm, D., M. A. Kay, et al. (2003). "Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6." Mol Ther 7(6): 839-850.
  3. J. Fraser Wright. Human Gene Therapy. July 2009, 20(7): 698-706. doi:10.1089/hum.2009.064.
  4. Yang Lu. Stem Cells and Development. February 2004, 13(1): 133-145. doi:10.1089/154732804773099335.
  5. Zincarelli, C., S. Soltys, et al. (2008). "Analysis of AAV Serotypes 1-9 Mediated Gene Expression and Tropism in Mice After Systemic Injection." Mol Ther 16(6): 1073-1080.
  6. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999;6:973–985.
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