10 Life Technologies
™
| Parkinson's cell models
Workflow for genome editing of iPSCs
The iPSC editing workflow we developed for this study
is shown in Figure 2A. First, feeder-free iPSCs were
electroporated with GeneArt
®
Precision TAL-FokI constructs
in the presence or absence of PCR fragments of donor DNA.
After electroporation, cells were immediately plated onto
mouse embryonic fibroblast (MEF) or Geltrex
®
matrix–coated
plates for recovery. The recovered colonies were picked and
screened for the presence of editing events. After subcloning
to create a homogeneous, clonal population that contained
a desired edit, isolated clones were further expanded and
characterized for pluripotency and genomic integrity.
Figure 2B shows the colony screening workflow and
the critical first steps of analysis of targeted genomic locations
in more detail. This part of the workflow is crucial for the
identification and isolation of a clonal population of edited
cells. Because most colonies recovered represented a
heterogeneous population of edited and unedited cells,
multiple rounds of colony screening steps were needed
to isolate clonal populations of edited cells.
A
iPSC culture
Electroporation
and recovery
Electroporation/
Recovery
Colony
screening
Colony
Screening
Clone
expansion and
characterization
Clone
Expansion/
Characterization
1–2 weeks 2–3 weeks 4–6 weeks
• Dissociated iPSCs
electroporated with
GeneArt
®
Precision
TALs and donor PCR
fragments using the
Neon
®
Transfection
System
• Cells plated onto
MEF or Geltrex
®
matrix–
coated plates
for recovery
• iPSCs cultured
in Essential 8
™
Medium
on Geltrex
®
matrix–
coated plates
• Cells dissociated
with StemPro
®
Accutase
®
Cell
Dissociation Reagent
• Colonies picked using
the EVOS
®
XL Imaging
System
• Colonies screened
using results from the
GeneArt
®
Genomic
Cleavage Detection Kit,
TaqMan
®
SNP
Genotyping Assay, or
Ion PGM
™
Sequencer
• Clones expanded
using KnockOut
™
iPSC
medium on MEFs or
Essential 8
™
Medium on
Geltrex
®
matrix
• Clones characterized
using pluripotent
marker antibodies,
TaqMan
®
hPSC
ScoreCard
™
Panel, and
the EVOS
®
FLoid
®
Cell
Imaging Station
