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Cell Biology JoVE (Open Access) Citable · DOI

Generation of Mice Derived from Induced Pluripotent Stem Cells

DOI: 10.3791/4003-v
What you'll learn
  • ✓ Reprogram mouse embryonic fibroblasts to iPSC lines using lentiviral vectors
  • ✓ Generate tetraploid blastocysts for iPSC integration and embryo development
  • ✓ Assess full pluripotency of iPSC lines by producing live offspring mice
Protocol

Biopharma Insights Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Difficulty
advanced
Total time
~3–4 months (iPSC derivation ~4 weeks; blastocyst generation and embryo transfer ~2–3 weeks)
Model organism
Mouse C57BL/6J
Biosafety
BSL-1

Steps

1
Prepare lentiviral reprogramming vectors

Generate lentiviral particles encoding pluripotency factors for transduction of somatic cells. This establishes the viral tool required for iPSC induction.

▶ 02:12
2
Derive iPSC lines from mouse embryonic fibroblasts

Transduce mouse embryonic fibroblasts with lentiviral reprogramming factors and culture to establish pluripotent stem cell lines. Select and expand colonies meeting pluripotency criteria.

▶ 03:24
3
Generate tetraploid embryonic blastocysts

Produce tetraploid blastocysts via electrofusion of two-cell embryos to serve as recipients for iPSC contribution to extraembryonic tissues.

▶ 04:37
4
Prepare iPSCs and inject into blastocysts

Culture iPSCs to optimal state, dissociate to single cells, and microinject into the tetraploid blastocyst inner cell mass cavity.

▶ 07:20
5
Analyze reprogramming efficiency and iPSC characterization

Assess lentiviral reprogramming outcomes, including iPSC colony morphology, pluripotency marker expression, and colony isolation efficiency.

▶ 09:20
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