Transcriptional analysis of the trapping cassette in an intron of pSPL3 vector.

(A) Potential splice variants I and II from the pSPL3-Trap(intron). Sequencing trace files representing the splicing of SD1 with SA and SA1 were shown, respectively. SD1 aSD1, splice donor for exon1; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SA2, splice acceptor for exon2. (B) RT-PCR analysis of transcripts from pSPL3-Trap(intron)-transfected HeLa cells. Sequencing results indicate the 1.2-kb band is derived from the splice variant I, which contains exon1, IRES and EGFP, and the 270 bp band results from splice variant II, which represents the proper spicing of exon1 with exon2 in pSPL3. (C) The absolute quantification of cDNA using real-time PCR was employed to determine the copy numbers of transcripts including EGFP (F+R, E = 96.1%, R² = 0.9981) and exon2 (F+R1, E = 97.2%, R² = 0.9989). Data are given as means ±standard deviation (n = 3). ** indicate P<0.01 versus the exon2 expression level in pSPL3-Trap(intron)-transfected cells.