It is essential to estimate the final titer for calculating the ideal MOI (number of viral particles per unit recipient cells). Statistical methods suggest that if all cells in a population are equally likely to be infected, and if 20% or fewer cells actually get infected then the probability of multiple insertions in each of the cells is very low.
However the MOI required to achieve 20% infection in different cell types varies enormously. For example, for mouse embryonic fibroblasts (MEFs) this can be achieved with an MOI of around 3 but for hESCs an MOI of greater than 20 is required.
■ For transduction of hESCs, concentrated virus is added directly to the culture medium of freshly plated hESCs. The cells are incubated with the virus for 24 h, after which the virus is washed off. Since the larger well spread feeders are more susceptible to viral infection, much higher amounts of virus are required to efficiently transduce hESCs cultured on MEFs. It is difficult to get infected hESCs at an MOI much less than 20.
■ Alternatively, the hESCs can be effectively transduced in suspension. This method not only eliminates the virus lost to MEFs but also increases the effective concentration of the virus, which results in increased infection of the hESCs for the same MOI.
1. After splitting, allow the hESC colonies to settle down in a 5 mL polystyrene tube.
2. Carefully aspirate most of the medium.
3. Resuspend cells in 400 pL of fresh complete medium containing the concentrated virus.
4. Incubate the cells in the tube at 37°C for 3-12 h. If viral titers are higher than 108 then suspension infection for as little as 1 h is sufficient. Longer incubations may cause the cells to aggregate.
5. Wash the cells by filling the tube with PBS and allowing the colonies to settle. Plate the infected cell clumps on Matrigel™-coated plates with MEFs in complete hESC medium.
■ Transduction efficiency is increased by including 5-10 ^g/mL polybrene (hexadimethrine bromide, Sigma H9268) in the transduction mix containing the cells and the virus.
■ eGFP expression in hESCs transduced with pSIN18.PGK-EGFP.WPRE vector can be first detected under a fluorescence microscope —24-48 h after transduction.
■ Transduced cells are typically assayed for eGFP expression (or for expression of transgene) by FACS 3-6 days after transduction.
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