Verifying the down-regulation of the endogenous genes during ES cell differentiation. (A) Diagrams are shown for the 5' portion of the novel embryonic isoform (upper, 2G2) as well as the most common isoform (lower, CSL) of CSL/RBP-Jkappa. The first exon (Exon N) of the novel embryonic isoform is located upstream of the second exon (Exon 2) which is common to all known isoforms. The predicted initiation codon "ATG" is indicated above the first exons and the primers (P1, P2, P3, P4 and P5) used in the semi-qauntitative PCR analysis below (Figure 5B) are also shown. "Int" stands for the retroviral integration site in the ES clone 2G2, which is located within the first intron of the novel embryonic isoform. Retroviral integration occurred in the same intron for two other ES clone 5C25 and 6B13. (B) Verification of the reduced expression of the novel early embryonic isoform of CSL/RBP-Jkappa in the differentiated cells (D) versus undifferentiated (U) cells by quantitative RT-PCR analysis. 1U and 2U, two independent total RNA samples from the undifferentiated wild-type ES cells. 1D and 2D, two independent total RNA samples from the differentiated cells. Equal amounts of total RNA was applied to every PCR reaction in each primer set. Four different primer sets (I, II, III, IV) were used in this quantitative PCR analysis. I, both primers (P1 and P2) are derived from the first exon unique to this novel isoform (Figure 5A). II, both primers (P3 and P4) are common to all the isoforms of CSL/RBP-Jkappa. III, the forward primer (P1) is derived from the first exon and the reverse primer (P4) is complementary in sequence to the second exon, which is common to all isoforms of CSL/RBP-Jkappa. IV, the forward primer (P5) corresponds to the first exon of the most common isoform of CSL/RBP-Jkappa and the reverse primer (P4) is complementary in sequence to the common second exon. "+RT" indicates that reverse transcriptase (RT) was included in the first-strand cDNA synthesis. "-RT" indicates negative controls in which no reverse transcriptase was added in the first-strand cDNA synthesis. (C) Confirming the down-regulation of the novel gene trapped in the ES clone 5C11 by Northern blot with a gene-specific probe. Total RNA samples were derived from three independent populations (Lanes 1, 2, 5) of the undifferentiated (U) wild-type ES cells and three independent populations (Lanes 3, 4, 6) of the differentiated (D) wild-type ES cells. The gel positions of 18S and 28S ribosomal RNA transcripts are marked with arrows.