Lentivirus general

Lentivirus has been widely used in molecular biology to introduce DNA into various cells. Due to the unusual ability to infect any host, lentivirus may be the only choice for certain cells.

I have been using a lentiviral system from Dr. Follenzi A. This system includes three packaging plasmids and one transfer plasmid. The three packaging plasmids are: pMDL, 8895 bp. Rev, 4174 bp. VSVG, 5824 bp.

The transfer plasmid I used is called 305. This construct features a PGK promoter driven partial NGFR as a stuffer which is followed by IRES and EGFP. Although very limited sites are available for cloning, 305 shows high efficiency of packaging, easy titration by FACS and some other advantages.

Similarly, I use another transfer plasmid 277 for ShRNA delivery. 277 contains PGK promoter driven EGFP. The biggest advantage of 207 is the small size which usually produce very high titer virus. As for ShRNA, I usually synthesize the short ShRNA sequence from OligoEngine and clone into pSUPER-hygromycin vector (from Oligoengine) and then cut the H1 promoter and ShRNA sequence out. After that, I ligate the H1-ShRNA fragment to 277 in a reverse orietation compared to PGK-EGFP.

The maximal titer I can get is about 10E6 (for 305) to 10E7 (for 277) virus/ml. I usually do not concentrate the virus.

I have been using lentivirus system to either deliver gene or ShRNA into adipocytes successively. Adipocytes are usually considered the most difficult cell for DNA transfection.


Comments

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Posted by neverthink (nevernetbug) Tue, 23 Dec 2008 15:44:01
Subject: More information you can find
For lenti/retro production and infections, I would recommend anyone getting into this approach to read Nolan\'s website at stanford(http://www.stanford.edu/group/nolan/)...It will get all the basics going. To go further, one might want to read a lot more or someone really experienced with it.... In any
case, for in vitro/cell culture, it is mostly easy and straightforward but might get challenging for some
in vivo work.

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