24 Life Technologies
™
| Parkinson's cell models
Because the α-synuclein protein was undetectable in all
of the iPSC lines, confirmation of SNCA knockout at the
protein level required differentiation of the lines into neural
stem cells prior to analysis by immunocytochemistry and
western blot. By both methods, the SNCA
+/–
line showed a
reduction of α-synuclein protein compared to the parental
line, and the protein was undetectable in the SNCA
–/–
line
(Figures 12A and B).
Figure 12. Confirmation of SNCA knockout. (A) Feeder-free iPSCs were differentiated to
neural stem cells (NSCs) using Gibco
®
PSC Neural Induction Medium. NSCs were fixed,
permeabilized, and stained for α-synuclein (green). Nuclear DNA was stained with DAPI
(blue). Images were captured with the EVOS
®
FLoid
®
Cell Imaging Station with the same
exposure, brightness, and contrast settings. (B) NSC pellets were resuspended in M-PER
Mammalian Protein Extraction Reagent. Equivalent amounts of protein for each sample
were loaded onto a Novex
®
10–20% Tris-Glycine Mini Protein Gel. Western blotting was
performed with primary antibodies (2 µg/mL α-Synuclein Monoclonal Antibody, mouse
(clone Syn 211) and 0.25 µg/mL GAPDH Mouse Monoclonal Antibody (clone 258)) diluted in
Membrane Blocking Solution and secondary antibody (2 µg/mL Goat Anti-Mouse IgG (H+L),
Alkaline Phosphatase Conjugate), incubated, and developed with BCIP/NBT.
Generation of α-synuclein knockout lines from the MSA line (continued)
Fluorescent images of the stained iPSCs and NSCs were taken with the EVOS
®
FLoid
®
Cell Imaging
Station. The three color channels allow for easy visualization of multiplexed staining and image overlay.
Learn more at lifetechnologies.com/floid
Instrument highlight—EVOS
®
FLoid
®
Cell Imaging Station
A
B
GAPDH
α-synuclein
1 2 3 4
1. Unedited (SNCA
+/+
)
2. Edited (SNCA
+/-
)
3. Edited (SNCA
-/-
)
4. Protein ladder
MSA parental MSA SNCA
+/-
clone 21 MSA SNCA
-/-
clone 32
