21
Life Technologies
™
| lifetechnologies.com/parkinsons
0.3
0.4
0.9
1.4
1.9
0.8 1.3 1.8 2.3 2.8
Mutant control
SNCA -/- detected
Het control
WT control
No template
Mutant
allele
WT allele
+
+
+
+
+
+
+
+
0.3
0.4
0.9
1.4
1.9
0.9 1.4 1.9 2.4
Mutant control
Clonal candidates
Het control
WT control
No template
Mutant
allele
WT allele
+
+
+
+
+
We used this same cell line containing a single copy
2 bp deletion in SNCA to generate a second line in which
both copies of the gene were disrupted. The iPSC clone
containing the single allele deletion was electroporated
again with the same TAL pair targeting SNCA, and the
colonies were screened after recovery. At this point, we
utilized a TaqMan
®
SNP Genotyping Assay for a fast and
efficient way of detecting allelic changes at a specific
location. A custom TaqMan
®
SNP Genotyping Assay was
designed to detect the 2 bp deletion and the wild type
sequence, and used to screen the colonies for double allele
deletion (Figure 9A). After identification of a colony that
contains a sequence lacking wild type SNCA, the identified
colony was subcloned, daughter colonies were analyzed,
and a clonal population of cells disrupted in both copies of
SNCA was identified (Figure 9B).
Figure 9. Screening for clones with SNCA double allele deletion by TaqMan
®
SNP Genotyping Assay. (A) Allelic discrimination plot, first round screening. As expected, most colonies
(green) from the primary screen were heterozygous (1 copy with no deletion; 1 copy with 2 bp deletion), but two colonies were homozygous (2 copies with 2 bp deletion; X's in circle). WT
control: template from the parental MSA line (2 copies with no deletion). Mutant control: template (from a donor plasmid) containing a 2 bp deletion in SNCA (2 copies with 2 bp deletion).
Het control: 50% unedited template/50% mutant template (1 copy with no deletion; 1 copy with 2 bp deletion). No template: TaqMan
®
reagents and water only. (B) Allelic discrimination plot,
second round screening of daughter colonies. The positive colony from the first round screening was dissociated and replated at a low density. The daughter colonies were screened with
the same SNP genotyping assay. Candidates of clonal populations with two mutant alleles are indicated as the blue dots on the left of the plot.
A B
Results
