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        Lentiviral Transduction Protocol for Human Embryonic Stem Cells

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        912

        实验试剂

         

        1.Puromycin Solution (Product No. P9620)
        2. Dispase II freshly prepared in media (Product No. D4693)
        3. Feeder Free hESC media
        4. DMEM/F-12 media
        5. Hexadimethrine bromide (Product No. H9268)

        实验设备

         

        1. 24-well culture dishes (Product No. CLS3526)

        2. Matrigel™ basement membrane

        实验材料

         

        MISSION® shRNA lentiviral particles

        实验步骤

         

        Prior to transduction — establish the Puromycin Kill Curve

        Determine the minimal puromycin concentration to kill all non-transduced cells. Use a range starting from 1–10 µg/ml of puromycin on the cells.

        Transduction Procedure
         

        1. DAY 0
         

        1)

        		  

        Passage cells when 80% confluent with large colonies approximately 24 hr prior to initial transduction using dispase at 1 mg/ml.
         

        2)

        		  

        Seed hESC cultures at a high density (1:3 is recommended) into a 24-well culture dish, taking care to maintain cells as aggregates of approximately 50–60 µm in diameter.

         

        2. DAY 1 (late afternoon )
         

        1)

        		  

        Feed hESC cells with 500 µl of pre-warmed (37 °C) mTeSR1 containing 6 µg/ml of polybrene (Hexadimethrine bromide).
         

        2)

        		  

        Replace cultures into incubator for 15 min.
         

        3)

        		  

        Add the desired amount of virus particles to the culture medium.
        NOTE: For the initial transduction of hESC lines, Dr. Cohen’s lab uses 10 µl of 1x106 TU/ml viral particles for H1 and H9 hESC lines. Since an exact cell count cannot be determined from the previous day’s plating due to extreme aggregation of the cells (ranging from 50 to 200 cells per clump), a precise MOI cannot be calculated. The most effective amount of virus particles to be added should be determined empirically for each cell line.

         

        4)

        		  

        Incubate for 18–20 hr at 37 °C, with 5% CO2 and 95% humidity.

         

        3. DAY 2 (mid-day )
         

        1)

        		  

        Remove medium, and replace with 500 µl of pre-warmed media supplemented with 6 µg/ml of polybrene.
         

        2)

        		  

        Replace cultures into incubator for 15 min.
         

        3)

        		  

        Add three times the initial amount (from Day 1) of viral particles to the culture medium.
        NOTE: Dr. Cohen’s lab uses 30 µl of 1x106 TU/ml of viral particles for the secondary infection.

         

        4)

        		  

        Incubate for 18–20 hrs at 37 °C, with 5% CO2 and 95% humidity.

         

        4. DAY 3 and 4 (morning )
         
         

        Remove medium daily and replace with 500 ml pre-warmed media without polybrene.

         

        5. DAY 5–8
         
         

        Remove media daily and replace with 500 µl pre-warmed media supplemented with puromycin. Dr. Cohen’s lab determined that a final concentration of 1 µg/ml puromycin worked best to select for successfully transduced H1 and H9 hESCs. Again, a puromycin kill curve should be run prior to any transduction experiment requiring puromycin selection.

         

        6. DAY 9

         
         

        To determine the efficacy of shRNA-mediated knockdown, carry out molecular assays to quantify mRNA and protein levels. Be sure to compare to appropriate controls.

        For subsequent analysis of additional time points, continue culturing hESCs with puromycin-supplemented media.

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