Figure 1.

PB Mutagenesis in OCT4 Reporter Human ES Cells

(A) Splicing consequences of PB[Insertional Mutagenesis,NANOG] (PB[IM,N]) insertion either in front of or within the intron of a gene. PB[IM,N] insertion into any reading frame is capable of inducing both overexpression of the downstream transcript with the β actin promoter and triple reading frame start cassette (not shown) as well as simultaneous expression of Katushka fluorescent marker by the splicing of Katushka/IRES into the native transcript with the splice donor (SD). This construct is also able to create N-terminally truncated transcripts with the splice acceptor, triple reading frame stop cassette, and poly A tail (SA-stop-pA). PB[IM,N] can also constitutively overexpress NANOG transcript from the independent NANOG overexpression cassette.

(B) (Top) FACS analysis of Katushka expression from PB[IM,N] in human ES cells 48 hr after transfection in single-cell suspension using the RFP channel. Mean of triplicate independent experiments is shown. Error bars represent SD. (Bottom) PB copy number within ten individual PB[IM,N] transfected human ES cell clones as assessed by real time PCR. There is a mean of five PB[IM,N] insertions per clone.

(C) Protocol of screen performed in H1 OCT4-EGFP cells for resistance to RA-induced differentiation using PB[IM,N]. Cells in six-well plates were transfected with PB[IM]N, puromycin selected, and subsequently treated with RA and G418. Cells were then split from the six-well plates into 10-cm plates at a 2:1 ratio and maintained on G418 alone.

(D) Brightfield and fluorescent images of the six unique clones recovered from the screen. All clones are positive for EGFP and Katushka fluorescent reporters for OCT4 expression and PB[IM,N] insertion within an actively expressed gene.