Home › Cell Biology › Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System
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Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

DOI: 10.3791/1447-v
What you'll learn
  • ✓ Transduce mouse embryonic fibroblasts with doxycycline-inducible lentiviral vectors
  • ✓ Induce pluripotent stem cell reprogramming via doxycycline-controlled transcription factors
  • ✓ Isolate and expand iPS cell colonies and verify pluripotency markers
  • ✓ Assess transduction efficiency and pluripotency using immunocytochemistry and flow cytometry
Protocol

Biopharma Insights The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Difficulty
advanced
Total time
~2–3 weeks (viral transduction day 1, doxycycline induction days 2–7, colony expansion and analysis days 8–21)
Model organism
Mouse embryonic fibroblasts (MEF)
Biosafety
BSL-2

Steps

1
Introduce lentiviral transduction to MEFs

Transduce mouse embryonic fibroblasts with doxycycline-inducible lentiviral vectors encoding Oct4, Sox2, Klf4, and c-Myc reprogramming factors. Incubate fibroblasts with virus-containing media to enable integration of transgenes.

▶ 00:09
2
Activate reprogramming with doxycycline treatment

Add doxycycline to transduced fibroblasts to induce expression of reprogramming transcription factors. Culture cells under induced conditions to promote pluripotent state conversion.

▶ 01:25
3
Analyze transduction efficiency by immunocytochemistry

Fix transduced cells and stain for transgene markers or viral integration markers to quantify the proportion of successfully transduced fibroblasts before reprogramming begins.

▶ 03:18
4
Isolate and expand iPS cell colonies

Manually pick emerging pluripotent stem cell colonies from culture plates and transfer to fresh feeder layers or defined media. Culture and expand colonies to obtain sufficient cell numbers for downstream analysis.

▶ 05:31
5
Verify pluripotency by immunocytochemistry staining

Stain expanded iPS colonies for pluripotency markers (e.g., Oct4, Sox2, Nanog) to confirm successful reprogramming to the pluripotent state.

▶ 07:39
6
Quantify pluripotency markers by flow cytometry

Perform flow cytometric analysis of iPS cells using antibodies against pluripotency-associated surface and intracellular markers to validate pluripotent identity.

▶ 09:14
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