Fig. 7. Transgenic Brn3a overexpression suppresses endogenous Brn3a transcription. (A) Structure of a transgene in which the 11 kb Brn3a sensory enhancer is used to drive the expression of a Brn3a cDNA transgene. The transgene product can be distinguished from the endogenous Brn3a gene by the presence of a Myc epitope sequence immediately after the transcriptional initiation site. (B-E) Expression of the Brn3a/Myc transgene in E13.5 embryos. In the wild-type embryos shown in B,D, Brn3a is detected in the dorsal root and trigeminal ganglia, and in specific groups of spinal cord and hindbrain interneurons (brackets). In the Brn3a knockout mice shown in C,E, only Brn3a expression from the sensory transgene is detected, and Brn3a is not expressed in the CNS. The arrows in E indicate signal originating from vascular artifacts. 5g, trigeminal ganglion; drg, dorsal root ganglion; HB, hindbrain; pit, pituitary. (F) Quantitative assays of the expression of the endogenous Brn3a message in isolated trigeminal ganglia from Brn3a wild-type and heterozygous E13.5 embryos in the presence and absence of the Brn3a/Myc transgene. In these assays, Brn3a mRNA encoded by the native locus was assayed selectively by the use of a 5'-PCR oligonucleotide that is interrupted by the Myc epitope sequence inserted into the transgene. Brn3a data are normalized to the expression of neuron-specific enolase (NSE). It is unlikely that the reduction in Brn3a expression observed in the presence of the Brn3a/Myc transgene is due to the presence of regulatory sequences in the transgene itself because in the transgenic line used, the transgene was present at only one or two copies, based on quantitative RT-PCR comparison with a single copy gene.