This is a ready-to-use second generation lentiviral vector preparation. The virus encodes firefly luciferase (Luc2) and human sodium iodide symporter (hNIS) cDNA under control of the spleen focus-forming virus (SFFV) promoter. The genes are linked via a P2A self-cleavage peptide.

The lentiviral vector is a VSV.G-pseudotyped HIV-1-based lentiviral vector that is capable of transducing a broad range of cell types and species. The SFFV promoter drives high constitutive expression in numerous cell types, and works very well in animal models.

The virus titer is functional infectious virus, not total virus particles, and indicates the number of transducing units (TU) per mL. The titer is determined using a WPRE probe-based qPCR assay that measures the number of copies of lentiviral vector stably integrated into the genome of transduced host cells. Unlike traditional p24 ELISA titrations, which measure both functional and non-functional lentiviral vector particles, this method measures the amount of functional lentiviral vector, which is more precise and relevant.

To increase biosafety, Imanis lentiviral vectors are self-inactivating (SIN). The viral enhancer and promoter have been deleted, which causes transcription inactivation of the LTR in the provirus; mobilization of replication competent viruses is prevented, while regulated expression of the genes occurs from the internal promoters without cis-acting effects from the LTR.¹

References:
¹Miyoshi et al. J Virol 1998. 72:8150-8157.

NIS Expression

HeLaH1 cells were transduced with LV-Luc2-P2A-hNIS at the indicated MOIs. After 1 week I-125 uptake by the cells was measured in the presence or absence of KClO4, an inhibitor of NIS-mediated I-125 uptake.

Luciferase Expression

HeLaH1 cells were transduced with LV-Luc2-P2A-hNIS (MOI = 10). To measure luciferase expression, 10⁴, 10⁵, or 10⁶ cells were placed in wells of a 96-well plate and 3 mg/mL of d-luciferin was added to the indicated wells. The plate was immediately imaged using the IVIS Spectrum imaging system.

Basic protocol

(Volumes are given for a 6-well plate; increase or decrease as needed.)
1. Seed cells in complete medium at an appropriate density to achieve 60-70% confluency at the time of transduction and incubate in an appropriate incubator.
2. Thaw the lentiviral vector stock on ice.
3. In a microcentrifuge tube, prepare virus at different MOIs (e.g. 1, 3, 10, and 30) in 1 mL total serum free media in the presence of Polybrene® (Imanis #REA001).
[Note: The final concentration of Polybrene® in the lentiviral vector mixture should be 4-8 ug/mL. Polybrene® can be cytotoxic to some cells and it is not advisable to incubate these cells with Polybrene® overnight; test cells in advance by incubating them with different concentrations of Polybrene®.]
4. Remove culture medium from cells and replace with prepared lentiviral vector.
5. Return cells to incubator.
6. After 3-4 hours, add 1 mL complete medium to each well and return to incubator.
[Note: if cells are sensitive to Polybrene®, the transduction mixture can be removed entirely and replaced with 2 mL of complete medium.]
7. 48-72 hours after transduction, check transgene expression according to an appropriate protocol.

Spin infection

(Volumes are given for a 6-well plate; increase or decrease as needed.)
1. Seed cells in complete medium at an appropriate density to achieve 60-70% confluency at the time of transduction and incubate in an appropriate incubator.
2. Thaw the lentiviral vector stock on ice.
3. In a microcentrifuge tube, prepare virus at different MOIs (e.g. 1, 3, 10, and 30) in 1 mL total serum free media in the presence of Polybrene® (Imanis #REA001).
[Note: Polybrene® is added to the transduction mixture to enhance transduction efficiency¹. The final concentration of Polybrene® in the transduction medium should be 4-8 ug/mL. Polybrene® can be cytotoxic to some cells and it is not advisable to incubate these cells with Polybrene® overnight; test cells in advance by incubating them with different concentrations of Polybrene®.]
4. Remove culture medium from cells and replace with prepared lentiviral vector.
5. Centrifuge the plate at 800 x g for 30-60 min.
6. Return cells to incubator.
7. After 3-4 hours, add 1 mL complete medium to each well and return to incubator.
[Note: if cells are sensitive to Polybrene® the transduction mixture can be removed entirely and replaced with 2 mL of complete medium.]
8. 48-72 hours after transduction, check transgene expression according to an appropriate protocol.

Transduction tips

1. Avoid repeated freeze-thaw cycles of the lentiviral vector, as this causes the titer of the virus to drop and decreases transduction efficiency.
2. The optimal MOI will vary between different cell types. Typically, primary cells require higher MOIs than established cell lines.
3. Transduction efficiency can be enhanced by including Polybrene® in the lentiviral vector mixture1, using a spin infection², and by reducing serum in the transduction mixture³.

References:
¹Konopka et al. J Gen Virol. 1991. 72:2685-2696.
²O’Doherty et al. J Virol. 2000. 74:10074-10080.
³Andreadis and Palsson. Human Gene Ther. 1997. 8:285-291.