Life Technologies
™
| Parkinson's cell models 20
To generate a clonal population of iPSCs with a 2 bp
deletion in codon 4 of the SNCA gene, Ion PGM
™
sequencing
was performed on selected colonies after the original
colony 21 was dissociated and replated. As shown in Figure
8A, no deletions were detected in the parental line, and the
original colony 21 identified as containing an editing event
was heterogeneous, having only 5% of cells showing the
2 bp deletion. After colony 21 was expanded, dissociated,
and replated, a colony that was enriched to 24% of the
sequencing events showing the 2 bp deletion was identified.
The stacked sequencing reads from the Integrative
Genomics Viewer (IGV) software for both the unedited
and edited cells is shown in Figure 8B. At this point, the
cells were dissociated, replated, and screened again, with
several colonies showing the 2 bp deletion in 50% of the
sequencing reads, indicating that a clonal population with a
heterozygous deletion was generated. Resequencing of the
population at a later passage confirmed a clonal population
with the same deletion.
Figure 8. Characterization of clonal populations of MSA iPSCs with a SNCA heterozygous deletion by Ion PGM
™
sequencing. (A) The percentage of bases called as deleted at the 2 bp
location increased as edited clones were enriched at each round of colony screening. (B) IGV software depicts stacked sequencing reads across exon 2 of the SNCA gene. The horizontal
black bars indicate locations within specific reads where a deletion was present. The 2 bp deletion in the fourth amino acid of SNCA is clearly distinguished when compared to the unedited
parental cell line.
Sample sequenced
Percent reads showing
2 bp deletion
Parental (wild type) 0
Edited pool (colony 21) 5
Daughter colony 24
Daughter colony after dissociation 50
Later passage 50
A B
Generation of α-synuclein knockout lines from the MSA line (continued)
SNCA
+/ -
edit – IGV viewer
Parental unedited clone
Edited SNCA
+/ -
clone
SNCA
+/-
edit as shown in IGV
Edited clone Parental unedited clone
SNCA
SNCA
