Using a recombinant adeno-associated viral (rAAV)-based gene-targeting method, we inserted the gene-encoding GFP into the SOX2 locus in H9 hESCs (Figure 1A). Proper homologous recombination led to the replacement of the SOX2 open reading frame with that of GFP and a neomycin selection cassette (SV40-Neo). After infection with rAAV and G418 drug selection, a total of 36 clones were expanded and screened by Southern blotting for homologous recombination events. Among these clones, 26 (72%) were found to carry the GFP-Neo cassette in the SOX2 locus (Figure S1A available online). No clones in which both SOX2 alleles were disrupted were isolated. Our subsequent analysis focused on one of these clones, clone 23 (hSOX2-23). We confirmed appropriate gene targeting in this clone using multiple restriction digests followed by Southern blotting (Figures 1B, S1B, and S1C). We did not observe nontargeted insertions of the rAAV sequences, and cells exhibited a normal karyotype (data not shown). Flow cytometry of hSOX2-23 revealed that the majority of the cells expressed GFP (Figure 1C). By comparison, a drug-selected clone, hSOX2-25, which was negative for targeted insertion (Figure S1A), showed no detectable GFP (Figure S2A). Despite only having one copy of SOX2, hSOX2-23 had similar levels of SOX2, OCT4, and NANOG expression as hSOX2-25 and wild-type (WT) hESCs (Figure S2B). Moreover, the percentage of GFP-positive (GFP+) cells in hSOX2-23 was constant over more than 20 passages. Immunofluorescence (IF) staining of hSOX2-23 showed that 100% of GFP+ cells expressed SOX2 protein (Figure S2C). Additionally, hSOX2-23 colonies had characteristic hESC morphology (Figure S2D) and expressed markers of the undifferentiated state, such as NANOG (Figure S2E). These results show that this rAAV-based gene-targeting method can be used to efficiently disrupt genes by homologous recombination. In addition, the SOX2-GFP hESC marker line can be used to monitor SOX2 expression in undifferentiated hESCs.
expanded and screened by Southern blotting for homologous recombination events
36 clones
clones
clones
26
clones
found to carry the GFP-Neo cassette in the SOX2 locus
72%
%
clones
one
hSOX2-23
percentage of GFP-positive (GFP+) cells
over more than 20 passages.
passages
GFP+ cells
expressed SOX2 protein
100%
%