Background: The adaptive immune system of vertebrates has an extraordinary potential to sense and neutralize foreign antigens entering the body. De novo evolution of genes implies that the genome itself expresses novel antigens from intergenic sequences which could cause a problem with this immune system. Peptides from these novel proteins could be presented by the major histocompatibility complex (MHC) receptors to the cell surface and would be recognized as foreign. The respective cells would then be attacked and destroyed, or would cause inflammatory responses. Hence, de novo expressed peptides have to be introduced to the immune system as being self-peptides to avoid such autoimmune reactions. The regulation of the distinction between self and non-self starts during embryonic development, but continues late into adulthood. It is mostly mediated by specialized cells in the thymus, but can also be conveyed in peripheral tissues, such as the lymph nodes and the spleen. The self-antigens need to be exposed to the reactive T-cells, which requires the expression of the genes in the respective tissues. Since the initial activation of a promotor for new intergenic transcription of a de novo gene could occur in any tissue, we should expect that the evolutionary establishment of a de novo gene in animals with an adaptive immune system should also involve expression in at least one of the tissues that confer self-recognition. Results: We have studied this question by analyzing the transcriptomes of multiple tissues from young mice in three closely related natural populations of the house mouse (M. m. domesticus). We find that new intergenic transcription occurs indeed mostly in only a single tissue. When a second tissue becomes involved, thymus and spleen are significantly overrepresented. Conclusions: We conclude that the inclusion of de novo transcripts in the processes for the induction of self-tolerance is indeed an important step in the evolution of functional de novo genes in vertebrates.