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Full SOP with Academic or a plan

Source Takahashi et al., 2006 · Institute for Frontier Medical Sciences, Kyoto University · 10.1016/j.cell.2006.07.024

Authors: Kazutoshi Takahashi, Shinya Yamanaka Duration: 42 days 8 phases Cell source: Mouse Embryonic Fibroblast (MEF); Mouse Tail-Tip Fibroblast (TTF)

Abstract

This protocol describes the generation of induced pluripotent stem (iPS) cells directly from mouse embryonic or adult fibroblasts by introducing four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4) under ES cell culture conditions. The resulting iPS cells exhibit morphology, gene expression profiles, and pluripotency equivalent to embryonic stem cells, as demonstrated by teratoma formation and embryonic contribution assays.

Cell source
Mouse Embryonic Fibroblast (MEF); Mouse Tail-Tip Fibroblast (TTF)
Application
Pluripotent stem cell generation and reprogramming

Protocol overview

48 steps across 8 phases

Phase 1 · MEF Isolation and Culture Preparation Day 0 to Day 7
  1. a Collect embryos from pregnant mice
  2. b Trypsin digest embryonic tissue
  3. c Neutralize trypsin and collect cells
  4. d Centrifuge and resuspend cells
  5. e Seed MEF cells for expansion
Phase 2 · Retroviral Vector Preparation and Transduction Day 1 to Day 5
  1. a Seed Plat-E packaging cells
  2. b Transfect Plat-E cells with retroviral vectors
  3. c Replace medium on Plat-E cells
  4. d Collect and filter viral supernatant
  5. e Infect MEF/TTF target cells with retrovirus
Phase 3 · G418 Selection and iPS Colony Formation Day 3 to Day 21
  1. a Add G418 selection antibiotic
  2. b Maintain G418 selection
  3. c Pick individual G418-resistant colonies
Phase 4 · Expansion and Characterization of iPS Clones Day 14 to Day 42+
  1. a Expand iPS clones on feeder layers
  2. b Assess colony morphology
  3. c Measure cell doubling time
  4. d Perform RT-PCR for ES cell marker genes
  5. e Perform alkaline phosphatase staining
  6. f Perform SSEA-1 immunostaining
Phase 5 · Teratoma Formation and In Vivo Pluripotency Assessment Day 28 onwards
  1. a Prepare iPS cells for injection
  2. b Anesthetize nude mice
  3. c Inject iPS cells subcutaneously
  4. d Monitor tumor growth
  5. e Harvest and fix tumors
  6. f Embed in paraffin and section
  7. g Perform hematoxylin and eosin staining
  8. h Perform immunohistochemical analysis
Phase 6 · In Vitro Differentiation and Embryoid Body Formation Day 14 onwards
  1. a Harvest iPS cells and initiate embryoid body formation
  2. b Culture embryoid bodies
  3. c Plate embryoid bodies on gelatin-coated dishes
  4. d Immunostain differentiated cultures for germ layer markers
  5. e Perform RT-PCR on differentiated cells
Phase 7 · Blastocyst Microinjection and Contribution Analysis Day 28 onwards
  1. a Prepare iPS cells for microinjection
  2. b Collect blastocysts
  3. c Microinject iPS cells into blastocyst cavity
  4. d Culture injected blastocysts
  5. e Transfer embryos into recipient females
  6. f Collect embryos at desired developmental stage
  7. g Perform immunohistochemistry on chimeric embryos
  8. h Allow embryos to develop to term (optional)
Phase 8 · Molecular and Genetic Characterization Day 14 onwards (parallel to other phases)
  1. a Extract genomic DNA for karyotyping
  2. b Perform Southern blot analysis of transgene integration
  3. c Perform bisulfite genomic sequencing
  4. d Perform chromatin immunoprecipitation (ChIP) assay
  5. e Perform Western blot analysis
  6. f Determine telomerase activity
  7. g Perform DNA microarray analysis
  8. h Perform PCR-based SSLP genotyping

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Attribution

This SOP was written by Organthis based on the published method in Takahashi et al., 2006. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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