Synphilin-1: generation of conditional knock-out mice
Synphilin-1 which was identified in yeast two-hybrid screens as an interacting partner of alpha-synuclein is a protein that is involved in the molecular pathogenesis of Parkinson's Disease (PD). Synphilin-1 is found in Lewy bodies in the brains of PD patients and it was shown to promote the formation of intracellular inclusions in cell culture where it co-localizes with alpha-synuclein. Additionally, a R621C mutation in the synphilin-1-gene was identified in two German PD patients. It is a substrate of parkin, dorfin and SIAH-1 and -2, all ubiquitin E3-ligases which are components of Lewy bodies. The neuronal expression of synphilin-1 and its colocalization in protein aggregates characteristic for neurodegenerative diseases suggest a more common role of synphilin-1 in the process of neurodegeneration. The physiological functions of synphilin-1 are still unknown. Dependant on alternative splice variants, neuroprotective and neurotoxic effects were described in cell culture models. To determine the effect of synphilin-1 deficiency in vivo, we have generated a new conditional mouse model. Using the pEasyFloxII vector, we have designed a gene targeting vector that contains exon 4 of the synphilin-1-gene flanked by loxP sites and two homologous arms which allowed a site specific recombination in transfected embryonic stem (ES) cells. After the identification of recombinant ES-cell-clones by neo-selection, PCR and Southern blot analysis, recombinant ES-cells were injected into blastocysts. Injected blastocysts were transferred to foster mice. The resulting chimeric mice were intercrossed with C57BL/6 mice. Heterozygous offsprings were mated with mice ubiquitously expressing the Cre-gene. Deletion of the exon 4 of synphilin-1 resulted in mice expressing only the alternative splice variant synphilin-1A known to be neurotoxic. The phenotypic consequences of this genetic mutation is being studied. We plan to study the effects of synphilin-1 deficiency in a genetic background of alpha-synuclein loss-of-function and gain-of-function mutations.