Despite the presence of scrapie associated fibrils in the brain tissue is characteristic ultrastructural for prion diseases, appropriate morphological structure of prions during the development of this disease is still unclear. The host prion protein (PrP) is encoded by the PrP gene (PRNP) found on chromosome 20 in humans and chromosome 2 in mice.
More recently, a novel correlative light and electron microscopy with Mini Singlet Oxygen Generator (miniSOG) is generated. MiniSOG, small protein 106 amino acids, Elisa kits can absorb blue light and emits green fluorescence was detected under a fluorescence microscope. MiniSOG can also catalyze the polymerization DAB portion to form a black-stained structures in the presence of osmium tetroxide, which can be observed under transmission electron microscope.
Two kind Recombinant proteins miniSOG-PrP expressing the resulting recombinant plasmid. photooxidation correlative and the transmission electron microscope is used to detect this plasmid.
Plasmids were microinjected into fertilized eggs FVB / NJ and Tg mice expressing miniSOG-PrP fusion protein was selected after a row raised withPRNP KO Tg mice.
Both strains of Tg mice, TgSOG23 and Tg231SOG, develop normally and maintained a healthy without abnormality is detected after one year of observation. Western blot and immunohistochemical test with the PRP and miniSOG specific antibodies confirms that the chimeric protein miniSOG-PrP expressed in brain tissue of Tg mice. Digital PCR tests suggest that the number of external gene copies inserted in TgSOG23 and Tg231SOG are 2 and 12, respectively.
Compared with the GFP tag miniSOG significantly smaller, which makes it easy to operate experiments and may have less influence on the biological function of a protein labeled. In addition, the GFP tag is an ideal marker for immunofluorescent tests, but may not be suitable for ultrastructural test for prion morphology.
Those Tg can supply new and useful animal models for further study on the formation of a potential morphological structures and deposits of prions in brain tissue during prion infection.