This protocol describes the testing of factors that can enhance the reprogramming of mouse embryonic fibroblasts (MEFs) to iPC cells using pMX-based retroviral expression.
Method Article
Reprogramming efficiency of mouse embryonic fibroblasts using pMX-based retroviral expression vectors: testing factors that enhance efficiency.
https://doi.org/10.1038/protex.2013.035
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This protocol describes the testing of factors that can enhance the reprogramming of mouse embryonic fibroblasts (MEFs) to iPC cells using pMX-based retroviral expression.
Note:
The success of this experiment is dependent on proper growth of MEFs and high retroviral titers. It is important to take all notes in this protocol into account.
Day 1
Note: for infections, 1 x 10 ml viral supernatant is sufficient for 40 infections of 250 ul. 1 plate delivers 2 x 10 ml viral sup for two rounds of infection.
Example:
2 x 10 cm plates for Oct4-pMX transfection
2 x 10 cm plates for Sox2-pMX transfection
2 x 10 cm plates for Klf4-pMX transfection
2 x 10 cm plates for c-Myc-pMX transfection
1 x 10 cm plate for each TAF-pMX transfection (12 plates to test TFIID-12).
Day 2
o 100 μl DMEM high glucose without additions (@RT)
o 2 μg pMX vector containing factor of interest
• don’t forget to include an empty pMX vector transfection to adjust for additions during infection.
o 2 μg pClEco packacking plasmid
o mix by flicking
o 12 μl Fugene HD (@RT) (Promega cat E2311/E2312)
o mix by flicking
o 15 min RT
o add to cells
Day 3
replace the medium of the transfected 293T cells from day 2 with 10 ml MEF medium/10 cm dish
Late afternoon: start up OG2 MEFs:
o plate Passage (P) 2 or 3 of OG2MEFs in MEF medium in 1 x 15 cm dish, gelatinized. Plate should be 70-80% confluent the next morning.
Note: the conditions of the MEFs and passage number are critical factors for successful reprogramming. For this reason, they are plated just before the start of the reprogramming experiment to keep the passage number as low as possible and the proliferation rate high.
Day 4
Note: to get an accurate number of healthy OG2 MEFS, it is advised to first plate the cells from a frozen vial and let them adapt for 1 night. Then, the next morning the appropriate number of cells can be plated in 6 wells plates.
o harvest the supernatant of the transfected 293T plates using a syringe and filter sterilize through a 0.45 μm low protein binding filter. This is the viral supernatant.
o Add fresh MEF medium (10 ml/10 cm dish) to the transfected 293T cells to allow a second harvest of viral supernatant after 24 hr.
o First prepare the infection mix containing all viral combinations in 15 ml tubes
• Use 250 μl viral supernatant per factor
• Add empty pMX viral supernatant when infecting with a lower number of factors to use the same amount of viral supernatant in each experiment
• Add 5 μg/ml final concentration protamine sulphate
• Infect in triplicate
• Example for infection of OSKM+ TFIID-12:
• Combine 250 μl viral supernatant of each of the OSKM reprogramming factors oct4, sox2, klf4, c-myc
• Add 250 μl of each of the TFIID-12 factors (TBP, TAF3, 4, 4b, 5, 6, 7, 9, 10, 11, 12, 13)
• Total volume in this case is 4 ml, mix gently before adding protamine sulfate
• Add 1:1000 from a 5 mg/ml stock solution of protamine sulphate (Sigma P4020)
o Replace the medium of OG2 MEFs with the infection mix. Use up to 4 ml infection mix per 1 well of a 6 wells plate.
Day 5
repeat the infection once more as described in day 4 using new viral supernatant that was formed in last 24 hrs.
discard the 293T cells
Note: the infected MEFs should clearly have grown with a confluency of ~25%. If this is not the case, the quality of the batch of MEFs used may not be optimal
Day 6
Note: the infected MEFs should clearly have grown with a confluency of ~50-70%
Day 7
Note: the infected MEFs should clearly have grown with a confluency of ~80%
Day 8
Note: the infected MEFs should clearly have grown with a confluency of ~90-95%
Note: this is important because the MEFs will proliferate and overgrow the plate. Without proper splitting, iPS cells will only appear at the edges of the plate and the results will not be reliable.
Day 20
Medium composition:
MEF medium
500 ml DMEM high glucose (Lonza BE 12-604F)
55 ml FBS (Hyclone cat SV30160.03)
5 ml Penicillin/Streptomycin (Lonza cat 17-603E)
5 ml glutamine (Lonza cat 17-605E)
ES cell medium
500 ml DMEM high glucose (Lonza BE 12-604F)
90 ml FBS (Hyclone cat SV30160.03)
6 ml Penicillin/Streptomycin (Lonza cat 17-603E)
6 ml Glutamine (Lonza cat 17-605E)
6 ml sodium pyruvate (Lonza BE 13-115E)
6 ml beta-mercaptoethanol (stock: 70 ul of beta mercapotethanol (Sigma M-6250) in 100 ml H2O, aliquot and store -20C)
6 ml non essential amino acids (lonza 13-114E)
60 ul LIF (500 U/ml final) (Gibco 13275-029)
20 days
Inclusion of TFIID-12 or TAF4 during OSKM-mediated reprogramming should result in a strong stimulation of the amount of iPS colonies and in the number of GFP+ cells (when OG2-MEFs were used). Both intermediate and strong GFP+ cells will be formed. The intermediate GFP+ cells represents a population of which many (~ half) will make it to the fully reprogrammed state upon prolonged culture.
We thank Hermann vom Bruch for technical assistance
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