Characterization of the Dlk2 transcription start site. A) Schematic exon-intron distribution of Dlk2 cDNAs putatively assigned to Dlk2. The arrows indicate the position of oligonucleotides used to specifically amplify Dlk2 transcripts. B) RT-PCR of Dlk2 transcripts with the following templates: cDNAs from 3T3-L1, NIH3T3, C3H10T1/2, or AT3F cells; cDNAs from heart (H), spleen (S), testicles (T), brain (B) and lung (L) of adult 129/C57BL6 mice; or genomic DNA (gDNA). Two PCR reactions were performed with each template, one to amplify variant 1 transcript (V1) and another to amplify variant 2 (V2). The sizes of the expected amplified DNA fragments are shown on the right. C) Analysis of Dlk2 mRNA expression levels in 3T3-L1, NIH3T3, C3H10T1/2 and AT3F cell lines by RT-qPCR. mRNA levels were referred to the expression level of phosphoriboprotein P0, which was used as an internal control. D) Experimental determination of Dlk2 TSS by 5' RACE. RNA from AT3F cells was used for 5' RACE amplification, using a specific reverse primer located within the sixth exon, at position +693 from the translation initiation codon (ATG), indicated by an arrow in the upper pannel. The amplified PCR products were cloned into the pCR2.1 vector, and twenty individual clones were sequenced. The 5' region sequences of genomic DNA and cDNA clone BC091431 are shown, including the 14 additional bases of the newly described clone FM180474.