Why choosing a lentiviral transduction as opposed to traditional lipid mediated plasmid transfection?
In order to track cells by optical imaging methods, we need to label them with light emitting reporters. Choices consist of bioluminescent or fluorescent tags. For bioluminescence we have the genetically encoded firefly luciferase, renilla and gaussia or bacterial luciferase. For fluorescence tags we have the genetically encoded fluorescent proteins such as GFP and RFP's or fluorescent dyes such as ICG, IRdye, Cy dyes, alexafluors and quantum dots, etc. (reagents page) Regardless of fluorescence vs. bioluminescence, a genetically encoded reporter has the significant advantage that if expressed under constitutive promoter, the signal is constitutively on and upon cell mitosis we do not have a dilution of signal as observed with dyes and qdots. The downside of using a genetic tag is the need for transfection. Traditional methods for transfection are lipid mediated plasmid transfections or electroporation. However the efficiency of these methods is low and it is laborious and time consumptive to obtain stable transfectants. A stable transfection as opposed to transient transfection is a fundamental requirement for longitudinal in vivo imaging. Over the past couple of years, multiple investigators have pursued the use of viral infection to establish stable integration of the foreign reporter DNA. Adeno-, retro and lenti-viral methods have been evaluated. When cells are retrovirally transduced, they are exposed to viral particles which contain the RNA and preformed enzymes such as RT and integrase etc. Keep in mind that the viral genome is not inside the engineered lentiviral particles (only the reporter gene). The viral genome (on a plasmid) is only present in the packaging cells. The viral genome consists of the gag-pol-env sequence and it is the pol part in which the RT is encoded.
Both retroviruses and lenti’s have RT in the viral particles. However: In contrast to oncoretroviruses, Lenti’s can productively infect non-dividing cells because the preintegration complex is actively transported through the nucleopore.
The Advantages of utilizing a Lentivirus are:
1) Ability to integrate the foreign DNA into non-dividing cells without silencing. (cancer cell lines, primary cell lines, white blood cells, stem cells, etc.)
2) Ability to infect cells from different species (human, murine, rat, monkey, etc.)
3) Self inactivating Long Terminal Reports (LTRs) in the lentiviral genome accommodate internal promoters, permitting Constitutive (CMV, ubiquitin C, etc.) as well as Inducible (vegfr2, NFkB, p53, etc) Reporter Systems.
4) The virus glycoprotein (VSVG) can be concentrated to achieve high titers (109 TU/ml).
References:
Kim et al., 2004 - Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Im Biol.
Bukrinsky et al., 1993 - A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature.
Links to Reporter Reagent Resources
