Down syndrome (DS) results from trisomy 21 (T21) and is the most frequent cause of cognitive impairment. Despite a worldwide effort to investigate the molecular mechanisms responsible for the DS phenotypes, the underlying causes are still poorly known. In this thesis, we aimed to better characterize the molecular mechanisms underlying the DS phenotypes. We showed how the use of isogenic models could improve our understanding of the DS molecular bases. We investigated the gene expression changes in DS using a pair of monozygotic twins discordant for T21. This unique model allowed the identification of transcriptome changes exclusively related to the T21. Additionally, we studied the chromosome 21 encoded Single minded 2 (Sim2) protein in order to better investigate the contribution of the chromosome 21 transcription factors in some of the DS manifestations. Altogether, the data provide new general insights to the understanding of the complex mechanisms causing the DS phenotypes.