Transporters for di- and tripeptides are found in prokaryotes and eukaryotes. In mammals two different transport systems have been identified. PEPT1 is mainly expressed in the small intestine and is responsible for the absorption of di- and tripeptides. PEPT2 shows a widespread expression within the organism. In kidney it mainly mediates the reabsorption of di- and tripeptides, but its role in other tissues such as brain or mammary gland has not yet been determined. Therefore, the aim of this work was to set the molecular basis for further studies on the physiological role of PEPT2 in particular by employing mice with targeted disruption of the Pept2 gene. First, the mouse PEPT2-cDNA was cloned by RACE. It is 3987 bp long and the coding region yields a protein of 729 amino acids identical to the other mammalian PEPT2 transporters. By Western Blot the apparent molecular weight of PEPT2 was around 100 kDa, suggesting that the protein is highly glycosylated. The PEPT2-mRNA was detected by Northern Blot in kidney, brain, mammary gland and lung. The functional characterization of the murine PEPT2 was performed in Xenopus laevis oocytes after injection of the corresponding PEPT2-cRNA. The carrier was found to transport di-, tripeptides and selected peptidomimetics in an electrogenic mode with a pronounced pH-dependency. Next, the Pept2 gene was isolated from a genomic DNA library from mouse. The Pept2 gene is 34-kb long and organized in 22 exons. Radiation hybrid analysis revealed the Pept2 gene to be localized on central mouse chromosome 16. Two major transcription start sites were identified by 5'-RACE, 35 and 235-bp upstream of the translation start. Moreover, the minimal promoter of the Pept2 gene was identified by reporter analysis and could be located between 432 and 286-bp upstream of the translation start and was found not to contain a TATA-box. Furthermore, a number of potential sites for transacting factors were identified in the upstream region of the Pept2 gene. These will be the basis of further studies on the regulation of the Pept2 gene. Based on the isolated Pept2 gene sequence it was possible to initiate the construction of a targeting vector for the generation of a Pept2 knock-out mice strain. The targeting vector was transfected by electroporation into mouse embryonic stem cells. After injection of homologe recombinant cells into blastocytes, those were transferred into pseudopregnant mice. One of the chimeric mice born showed germline transmission leading to heterozygote Pept2 knock-out mice that will allow now to establish a Pept2-/--mouse strain for studies on the physiological role of the peptide transporter PEPT2 in mammalian organisms.     «

Transporters for di- and tripeptides are found in prokaryotes and eukaryotes. In mammals two different transport systems have been identified. PEPT1 is mainly expressed in the small intestine and is responsible for the absorption of di- and tripeptides. PEPT2 shows a widespread expression within the organism. In kidney it mainly mediates the reabsorption of di- and tripeptides, but its role in other tissues such as brain or mammary gland has not yet been determined. Therefore, the aim of this wo...     »