11
Life Technologies
™
| lifetechnologies.com/parkinsons
Figure 2. Workflow for genome editing of iPSCs. (A) Feeder-free iPSCs cultured in Essential 8
™
Medium and on Geltrex
®
matrix–coated plates were treated with thiazovivin before
dissociation with StemPro
®
Accutase
®
Cell Dissociation Reagent. Dissociated cells were electroporated with TAL constructs with or without donor DNA using the Neon
®
Transfection
System, and then plated at a low density onto MEF or Geltrex
®
matrix–coated plates for recovery. After 1 to 2 weeks, colonies were picked, screened, and selected based on results obtained
with the GeneArt
®
Genomic Cleavage Detection Kit, TaqMan
®
SNP Genotyping Assay, or Ion PGM
™
Sequencer. The final clones were expanded and characterized to confirm pluripotency
and genomic integrity. (B) Colony screening workflow. Colonies were manually divided in half, with half the cells analyzed and the other half cultured until analysis was complete. PCR
amplification of genomic DNA was performed to obtain the sensitivity needed to monitor changes at the target location in the GeneArt
®
Genomic Cleavage Detection and PGM assays.
These resulting amplicons were then analyzed using the three screening methodologies shown. Note that the sensitivity of the TaqMan
®
SNP Genotyping Assays is such that the PCR steps
can be omitted if the other methodologies are not used.
Treat iPSCs with StemPro
®
Accutase
®
to dissociate, then
transfect with TAL-FokI pairs
Plate cells at low density to form
well-separated colonies
Allow 1–2 weeks for colony
expansion, then harvest colonies
using pipettor
Each colony half is kept for
analysis, half is expanded
Colonies containing editing events
dissociated using Accutase
®
reagent, plated at low density,
and screening process repeated
to isolate a clonal population
Harvest cells for
analysis
Extract genomic DNA
Perform 1
st
round of
PCR amplification
Perform 2
nd
nested
PCR amplification
TaqMan
®
SNP
Genotyping
GeneArt
®
Genomic
Cleavage
Detection
Ion PGM
™
Sequencing
Analysis sub-workflow
B
Methods
Analysis Expansion
★
Analysis Expansion
★
