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Genetics / Genomics JoVE (Open Access) Citable · DOI

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

DOI: 10.3791/54718-v
What you'll learn
  • ✓ Perform RMCE-based gene editing at the AAVS1 locus in human pluripotent stem cells
  • ✓ Execute nucleofection transfection and antibiotic selection protocols for hPSC engineering
  • ✓ Generate and characterize isogenic hPSC lines for comparative transgenic studies
Protocol

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Difficulty
advanced
Total time
~7–10 days (transfection to clonal expansion and characterization)
Model organism
Human pluripotent stem cells (hPSCs)
Biosafety
BSL-1

Steps

1
Prepare FRT-containing hPSC line for transfection

Culture and ready the FRT-flanked hPSC line and prepare the recombinase expression construct for nucleofection. Ensure cells are at appropriate confluence and viability.

▶ 00:58
2
Perform nucleofection of hPSCs with RMCE vector

Transfect the prepared hPSC line using nucleofection technology to introduce the recombinase-containing cassette. Optimize parameters for maximum transfection efficiency.

▶ 04:44
3
Apply positive and negative selection to edited cells

Use antibiotic selection markers to enrich for cells successfully undergoing recombinase-mediated cassette exchange. Eliminate non-recombined and intermediate clones.

▶ 06:28
4
Expand and characterize RMCE-derived hPSC lines

Culture selected clones to sufficient cell numbers and perform molecular/functional characterization to confirm proper editing, pluripotency, and isogenic status.

▶ 07:28
5
Validate RMCE efficiency and transgene integration

Analyze results including integration patterns, transgene expression, genomic integrity, and efficiency metrics. Confirm suitability for downstream comparative studies.

▶ 08:31
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