Protocol for Ready-to-Use Lentivirus Markers/Supernatants

Product Description

Expressing a marker in specific cells is a powerful tool to monitor or sort out a pre-labeled cell if differentiated to desired cells in mammalian species. Biogenova’s Ready-to-use product lines have packed mark gene to overexpress in vitro and vivo. These generated mark genes enable scientists to trace any stages of cell differentiation.

Unlike murine-based MMLV or MSCV retroviral systems, our lentiviral-based particles permit efficient infection and integration of the construct into differentiated and non-dividing cells, such as cardiomyocytes, neurons and dendritic cells, overcoming low transfection and integration difficulties when using these cell lines. Self-inactivating replication incompetent viral particles are produced in packaging cells (293T) by co-transfection with compatible packaging plasmids. In addition, the Transduction Particles are pseudotyped with an envelope G glycoprotein from Vesicular Stomatitis Virus (VSV-G), allowing transduction of a wide variety of mammalian cells. Because VSV-G over-expresses extra envelope glycoprotein which can be attached on the cells to protect viral particle to infect cells in lentivirus packaging procedure, Biogenova not only offers packaged high titer lentivirus but also a highly purified lentivirus to easily and effectively transduce any desired cells. It has additional potential to generate transgenic mouse with specific tissue promoter.

Components/Reagents

The Lenti-Green etc. are provided as a frozen stock containing 5x10⁶-10⁷ lentiviral Transducing particles per ml in Dulbecco’s Modified Eagle’s Medium with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin.

Precautions and Disclaimer

These products are for R&D use only, not for drug, household, or other uses. Though the lentiviral transduction particles produced are replication incompetent, it is highly recommended that they be treated as Risk Group Level 2 (RGL-2) organisms. Follow all published RGL-2 guidelines for handing and waste decontamination. Also, use extra caution when using lentiviral transduction particles that express GFP etc., involved in cell differentiation (such as embryo stem cell).

Storage/Stability

All components are stable for at least six months after receipt when stored at -70ºC. Repeated freeze/thaw cycles are to be avoided, as this will severely reduce transduction efficiency.

Procedure for Suspension Cells

Various protocols have been used for transducing suspension cells. We recommend transducing suspension cells with our Ready-to-Use lentivirus in a small volume and gently centrifuging the cells through the supernatant. Here we give an example of transducing cells by centrifugation in a 6-well plate, cells in tissue culture flasks can be harvested in a 15ml conical tube and follow the same procedures.

1. Day 1: Plate the mammalian cell line of choice in complete medium 24 hours prior to transduction at 5 x 10⁵/ml in a well of a 6-well plate (use 2 ml/well).
    
2. Day 2: Thaw the lentiviral stock at room temperature. Add lentiviral supernatant at 1:1 ratio of cell culture medium.
   
   Note: You Do Not need to add polybrane for viral transduction. Our products already contain the appropriate concentration of Polybrane for a 1:1 dilution in the cell culture medium. If you use a different ratio, such as 1:10, you need to add more polybrane accordingly (we recommend a final conc. of 8 ug/ml).
   
   1. When transducing a lentiviral construct into a cell line for first time, it is recommended that a range of MOIs (such as 0, 1, 10 and 50) be used to determine the optimal degree of mark gene expression.
   2. When overnight incubation presents a toxicity concern, cells may be incubated for as short as 4 hours before changing the medium.
   3. Spin the plate at 3000 x g for 4 hrs at 30°C (Please note: This procedure is necessary only for suspension cells). After spin, take out medium containing viruses and re-suspend cells in fresh complete medium. Return cells to 37°C/CO₂ incubator for 12 hrs or overnight. The same procedure can be repeated to increase transduction rate.
      
3. Day 3: Remove the old medium and replace it with fresh, complete culture medium and keep to incubate overnight.
   
4. Day 4: Check fluorescence which will be detectable with an appropriate wavelength under fluorescent Microscope.

Procedure for Adherent Cells

1. Day 1: Plate the mammalian cell line of choice in complete medium 24 hours prior to transduction at 5 x 10⁵ml in a well of a 6-well plate (use 2 ml/well).
   
2. Day 2: Thaw the lentiviral stock at room temperature and dilute (if necessary) the virus to a suitable MOI (multiplicity of infection) with fresh complete medium. (The volume of stock viral supernatant can be 1:10 to 1:1 of the volume of medium which will be used to culture the target cells). Remove the culture medium from the cells. Add fresh medium containing virus.
   
   Note: You Do Not need to add polybrane for cell transduction. Our products already contain the appropriate concentration of Polybrane for a 1:1 dilution in the cell culture medium. If you use a different ratio, such as 1:10, you need to add more polybrane accordingly (we recommend a final conc. of 8 ug/ml).
   
3. Day 3: Remove the medium containing virus and replace with fresh and complete culture medium.
   
4. Day 4: Check fluorescence which will be detectable with an appropriate wavelength under fluorescent microscope.

References

1. Zufferey, R., et al., Multiply attenuated lentiviral vector achieves efficient gene delivery in
vivo. Nat. Biotechnol. 15, 871-85 (1997).

2. Zufferey, R., et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene
delivery. J Virol. 72, 9873-80 (1998).

3. Burns, J.C., et al., Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors:
Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA, 90, 8033-8037 (1993).

4. NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) 2002 (http://www4.od.nih.gov/oba) Polybrene is a registered trademark of Abbott Laboratories Corporation.

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