The identities of the trapped genes were verified by RT-PCR analysis of the fusion transcripts. (A) RT-PCR products of the four candidate ES clones (2G2, 5C25, 5C11 and 5C1). The primer Xho-Junc (P1), which has a sequence complementary to the junction of the SA site and I(N) sequence in the gene trap vector eGeoN/E+pA, was used for reverse transcription in the first-strand cDNA synthesis. Another primer Bcl-2R2 (P2) complementary to exon 3 of Bcl-2 in eGeoN/E+pA was paired with the gene-specific primer (P3) to amplify the cDNAs derived from the fusion transcripts between the endogenous trapped genes and the gene trap vector. The positions of these primers (P1, P2, P3) with respect to the gene trap vector and the trapped endogenous genes are indicated in Figure 4B and Figure 4C. Arrows indicate the agarose gel positions of the expected RT-PCR products. "+", reverse transcriptase (RT) was included in the first-strand cDNA synthesis (Lanes 2, 4, 6, 8). "-", no reverse transcriptase was added to the first-strand cDNA synthesis (Lanes 3, 5, 7, 9). Lane 1 and Lane 11, 1kb plus DNA ladder (Invitrogen/Gibco). (B) Schematic diagrams illustrating the fusion transcripts expressed in the trapped ES clones when the gene trap vector is inserted into an intron of the active endogenous genes (e.g. ES clone 2G2 and 5C25). As shown in the diagrams, the vector is inserted into the Intron N between Exon N and Exon N+1. After the splicing of the fusion transcripts, the splice donor present at the junction of Exon N/Intron N was utilized to join the Exon N directly to the splice acceptor (SA) present in front of the GFP and Neo reporter genes. (C) Schematic diagrams illustrating the fusion transcripts expressed in the trapped ES clones when the gene trap vector is integrated into an exon of the active endogenous genes (e. g. ES clone 5C1, the first exon of Zfp-57). As shown in the diagrams, the vector is integrated into Exon N, which results in the split of Exon N into two parts (Exon N-5' and Exon N-3'). A read-through fusion transcript will be generated or the cryptic splice donor site present in the 3'LTR of the vector is utilized to join the Exon N-5' part and portion of the 3'LTR directly to the splice acceptor (SA) present in front of the GFP and Neo reporter genes.