Successful Transduction Using Lentivirus

Methods and tips for successful use of lentivirus products.

Successful targeting relies on optimizing key sensitive steps in the process, including lentiviral transduction. Below are some helpful handling and titration tips from our R&D lentiviral experts. As always, contact our technical support if you have any questions about your genome modification project at CRISPR@sial.com.

• Overview of Lentivirus Transduction protocol
  

Definition of key terms:

MOI - Multiplicity Of Infection - The ratio of the number of transducing lentiviral particles to the number of cells.
VP - Viral Particle - the extracellular infective form of a virus, consisting of an RNA or DNA core inside a protein envelope
TU - Transducing Unit - the number of functional viral particles in a solution that are capable of transducing a cell and expressing the transgene

Handling Lentivirus:

• Lentiviruses are sensitive to extreme temperature shifts. Multiple freeze-thaw cycles and prolonged exposure to ambient temperatures will decrease the functional lentiviral titer.
  
  
• Thaw lentiviral particles on wet ice. Keep them stored on ice while performing lentiviral transductions.
  
  
• Lentivirus should be aliquoted into smaller working volumes upon first thaw. Return all aliquots not used in your initial experiment immediately to -70° C. Alternatively, order your lentivirus in working volume aliquots and only thaw the volumes you will need for your transductions.
  
  
• Soak all virus and any techware and tips that touch virus or virus-containing cells in 10% bleach for 20 minutes before discarding into biohazardous waste.

Recommendations and steps to optimize your experiment setup

• Establish a kill curve for each cell line you will use to identify optimal antibiotic concentration needed to select for lentiviral-integrated cells
  
  
• Determine functional titer for each cell line and lentiviral vector combination you will use (see below)
  
  
• Determine Multiplicity of Infection (MOI) for each cell line and lentiviral vector combination you will use. MOI is the ratio of the number of transducing lentiviral particles to the number of cells. It is highly recommended that for each new cell type to be transduced, a range of MOI be tested.
  
  
  • Plate 1.6 x 10⁴ cells into wells of a 96-well plate with 120 µL fresh media.
  • Add control virus to cells in a range of MOIs. For most cell types, a range of 0.1 - 10 MOI is suitable. For hard to transfect cell lines you may need to increase your range to MOI of 50 or 100.
  • If using antibiotic selection: apply selection media and identify well with viable cells at the lowest tested MOI value. This is the MOI to use in your future transduction experiments for this cell line and vector
  • If using fluorescence: identify the well with desired quantifiable fluorophore expression at the lowest tested MOI value.
    
• To calculate the amount of virus needed after establishing MOI:
  • (total number of cells per vessel or well) x (desired MOI) = total TU needed
    
  • (total TU needed) / (TU/mL functional titer) = total mL of lentiviral particles to add to each well

Titering Lentivirus Particles:

After a lentivirus preparation is complete, the titer will need to be determined prior to transduction. Viral titers reported for our lentiviral products are determined by p24 ELISA. This assay measures lentivirus-associated p24 capsid protein.

• We determine the TU/mL based upon the p24 assay from ZeptoMetrix. We use the conversion from Didier Trono to determine the relationship between pg/mL p24 and viral titer:
  
  
• Known variables:
  1. There are approximately 2000 molecules of p24 protein / physical particle (PP) of lentivirus.
  2. 1 Da = 1 g/mol
  3. Avogadro's Number = 6 x 10²³ molecules/mol
  4. The molecular weight of p24 protein is 24 x 10³ Da = 24 x 10³ g/mol
  
  
• Equation:
  (2000 molecules/PP) ● (24 x 10³ g/mol)= 48 x 10⁶ g ● molecule / PP ● mol
  
  (48 x 10⁶ g ● molecule / PP ● mol) ÷ (Avogadro's Number) =
  (48 x 10⁶ g ● molecule / PP ● mol) ÷ (6 x 10²³ molecules/mol) = 8 x 10^-17 g/PP
  
  With rounding, 8 x 10^-17 g/PP ≃ 1 x 10^-16 g/PP
  
  Convert g/PP to pg/PP: (1 x 10^-16 g/PP) ● (1 x 10¹² pg/g) = 1 x 10^-4 pg/PP
  
  Find the inverse:
  If 1 x 10^-4 pg = 1 PP; the inverse is 1 pg = 1 x 10⁴ PP
  
  Therefore, 1 pg of p24 protein contains approximately 10,000 PP

The efficiency of packaging a delivery construct, also known as the transfer vector, into lentiviral particles can vary greatly depending on the size and composition of the transfer vector. Thus, a reasonably well packaged, VSV-G pseudotyped lentiviral vector will have an infectivity in the range of 1 TU per 200 viral particles for inefficient transfer vectors to 1 TU per 1 viral particle when 100% efficient viral packaging occurs. It is important to note that measuring the functional titer of a recombinant lentivirus is not only dependent on how efficiently the transfer vector is packaged but also the transduction efficiency of the cell line used.

• Custom and off the shelf lentivirus preparations are provided with a certificate of analysis (C of A) that indicates the p24 titer in transducing units/mL (TU/mL).
  
  
• When produced via 96-well format (small scale), our lenti vectors have been characterized to yield between 1x10⁶ and 1x10⁷ TU/mL (as measured by the p24 ELISA).
  
  
• Higher titers and volumes (large scale) are available. Available titers range between 1x10⁷ and 1x10¹⁰ TU/mL (as measured by p24 ELISA) and volume options range from 0.1 mL to 10 mL. Note: not all volume options are available in the highest titers.
  
  
• The correlation between p24 and functional titer is vector and cell-line specific. At the outset of each transduction experiment, first determine the relationship between p24 titer and functional titer of each vector in the cell line you will be using. Common methods for determining functional titer include:
  • Limiting dilution protocol to generate colony forming units (CFU assay)
  • Fluorescence-Activated Cell Sorting (FACS) Titration
  • qRT-PCR
  • Visit Lentivirus Protocols page for details
    
• We offer a variety of off-the-shelf controls and custom-made controls designed to optimize experimental setup and determine the relationship between p24 titer and functional titer. Once the correlation between p24 and functional titer for each cell line(s) and vector(s) is determined, the same correlation can be used for remaining experiments with the same vector and cell line.