Figure 1
From: A platform for rapid generation of single and multiplexed reporters in human iPSC lines
Efficient targeting of Chr. 19 and Chr. 13 safe harbor loci.
The experimental strategy of generating AAVS1-copGFP (a) and Chr13-copGFP (b) iPSC lines. Solid black triangles represent the loxP sites and triangles filled with diagonal lines represent Lox sites for RMCE. Testing primer sets for “Left” (Left arm integration test), “Right” (Right arm integration test) and “ORF” (WT ORF test) were also illustrated. (c) Validation of AAVS1-copGFP heterozyte and homozygote clones by junction PCR (upper) and sequencing (lower). (d) Validation of Chr13-copGFP heterozygote clone by junction PCR (upper) and sequencing (lower). (e) Quantification of donor vector copy number via qPCR. Data were presented as %mean control using the previously validated single copy line “CAG-GFP-Chr19” as the 100% control. (f) The copGFP reporter gene in AAVS1-copGFP line was not silenced while differentiated into NSC. Nestin (red) antibody was used to label NSC. Scale bar is 100 μm. (g) The copGFP reporter gene in Chr13-copGFP line was not silenced while differentiated into NSC. Scale bar is 100 μm. (h) FACS analysis of GFP-positive cells in a representative culture (Chr13-copGFP NSC) (right panel) and the non-labeled WT control NSC (left panel).
