By Paul J. Verma
This quantity offers an figuring out of the criteria keen on nuclear reprogramming, that's crucial for the luck of reprogramming. The booklet is geared toward reprogramming differentiated cells and germ line transmission of pluripotent stem cells and lines chapters that care for reprogramming-related concerns akin to research of mitochondrial DNA in reprogrammed cells and the isolation of reprogramming intermediates; substitute tools for nuclear move; the construction of germ-line chimeras from embryonic stem cells and brought on pluripotent stem cells; and neonatal care and administration of somatic cellphone nuclear move derived offspring. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols and tips about troubleshooting and heading off recognized pitfalls.
Authoritative and state-of-the-art,
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Additional resources for Cell Reprogramming: Methods and Protocols
BJ fibroblasts were transduced with Sendai virus overnight and incubated in standard fibroblast media for 7 days. Seven days post-transduction, cells were transferred to feeder-free condition. Colonies were stained with Tra1-60 antibody on the 28th day post-transduction Fig. 2 Generation of iPSCs on xeno-free condition. BJ fibroblasts were transduced with Sendai virus overnight and incubated in standard fibroblast media for 7 days. Seven days post-transduction, cells were transferred to xeno-free conditions.
7. 5 × 105 cells per well of a 6-well plate in a total volume of 2 mL of human fibroblast medium per well. 1 “Generating conditioned Pluriton medium”). 8. If reprogramming under low oxygen conditions, the medium should be equilibrated at low O2 tensions. 9. Do not leave the mRNA transfection complex in the culture medium for longer than 4 h, as prolonged exposure to the Synthetic mRNA Reprogramming of Human Fibroblast Cells 27 RNAiMAX transfection reagent will result in increased cellular toxicity.
Prepare MEF feeder cells in 12-well plates 1 day before iPSC colony pickup. 2. Thaw one aliquot of Pluriton supplement on ice and add 4 μL of the supplement to 10 mL of Pluriton medium to generate Pluriton reprogramming medium. 3. Aspirate the MEF culture medium from 12-well MEF feeder plates. 4. Add 1 mL of PBS to each well to rinse and aspirate the PBS. Synthetic mRNA Reprogramming of Human Fibroblast Cells 25 5. Add 1 mL of human iPSC culture medium to each of the rinsed wells. 6. Aspirate the medium from the well of the 6-well plate that the primary iPSCs will be picked from.