Establishing Transgenic Mice Overexpressing Shadoo Protein And Studying About Shadoo Protein Pathogenesis In Prions

The protein was coined Shadoo and the gene was termed Sprn. Sprn seems tobe widely conserved in nature, being present in the genomes of lower organisms suchas zebrafish all the way up to rodents and primates. Sprn can be found onchromosomes7and10in mice and humans, respectively. Like Prnp and Prnd, theentire open reading frame of Sprn is contained within a single exon. Bioinformaticanalysis of Sprn and Prnp sequences from diverse organisms has suggested that theprimeval prion protein gene may have been related to Sprn, with the Prnp and Prndgenes evolving later. Murine Shadoo is predicted to be a98residue protein with N-and C-terminal signal sequences mirroring those in PrP~Cand Doppel. Shadoo N-glycosylation is also conserved, but is only predicted to occur at a single site.Although Shadoo lacks the octarepeat sequences found in PrP~C, it does have a seriesof N-terminal charged tetrarepeats rich in glycine, serine, alanine and arginine. Asmentioned, the bulk of the homology between Shadoo and PrP~Cis found within thehydrophobic tract. Murine Shadoo is a GPI-anchored cell-surface glycoprotein whichundergoes endoproteolysis in a manner reminiscent of PrP~C.Prion disease is the prions (PrPSc) cause a kind of infectious, pathogenicNeurodegenerative disease, PrPScis normal PrP~Cthe change of conformation.Although prion disease research many important achievements, but so far It is notclear that the pathogenesis from PrP~Cto PrPSc. Study found that in the process ofchange to PrP~Cto PrPSchost body has "protein X" play a decisive role, this kind"protein X" may be will help people to understand the pathogenesis of prion disease.In recent years, Scientists found novel prion family members-the third Shadoo proteinthrough comparative genomics, it is people need to search for "protein X". This paperhope that through establishing overexpressing Shadoo protein transgenic mice model,the mice model were inoculated mouse-adapted Scrapie22L, analysis Shadoo proteinon the role of prions disease, this paper discussed the mechanism of prions disease.The study were composed by two parts:1. The construct of Shadoo overexpressing transgenic mice lines.1) Establishment of sprn transgenic mice andobtained three positive founder mice, respectively, named TgMoSprn-D, TgMoSprn-B, TgMoSprn-K.2) Sprn transgenic mice carrying exogenous gene expression.3)The transgenic vector under control of the PrP promoter, Shadoo protein expressionin brain, thereby mirroring expression of endogenous PrP.4)Overexpression ofShadoo in the brains of mice from the three lines TgMoSprn-D, TgMoSprn-B andTgMoSprn-K was confirmed by Western blotting,Compared to wild-type mice,respectively, is6.20,4.51and2.83times.5) Immunohistochemitry detection ofShadoo protein overexpression in pyramidal cells cerebral cortical cells and Purkinje'scell of transgenic mice.2. The pathogenesis of overexpressing Shadoo proteintransgenic mice inoculated with mouse-adapted22L scrapie.1)There are differentbrain pathological lesions in wild-type mice and transgenic mice, in our Shadootransgenic mice, we also detected large amyloid plaques not seen in wild-type mice.2)There are different PrPScdeposits in transgenic mice and wild-type mice. We haveobserved small punctate PrPScdeposits in wild-type mice and large nonflorid PrPplaques deposits in transgenic mice.3) In transgenic mice, overexpressing Shadooprotein does not change expression of the PrPc and does not affect expression ofPrPScinoculated with scrapie, and transgenic mice and wild-type mice has a similarproportion of glycosylation.4) overexpressing Shadoo protein did not change thesurvival time and the survival rate of transgenic mice after inoculated with22Lscrapie.In conclusion,1) This study has successfully established overexpressing Shadooprotein transgenic mice line, and successfully bred three different Shadoo proteinexpression transgenic mice line, and the Shadoo prion protein expression patternsmirrored expression of endogenous PrP.2) In our Shadoo transgenic mice, we alsodetected large amyloid plaques not seen in wild-type mice. We have observed smallpunctate PrPsc deposits in wild-type mice and large nonflorid PrP plaques deposits intransgenic mice.3) Overexpressing Shadoo protein did not change the expression ofendogenous PrP~Cin transgnic mice and did not affect the amount of proliferationPrPScin transgenic mice after inoculated with scrapie, further demonstrating thatShadoo protein and PrP protein are independent in the prion pathogenesis.4)Overexpressing Shadoo protein did not change the survival time and the survival rate of transgenic mice after inoculated with mouse-adaptd22L scrapie, implying thatShadoo overexpression did not have any additive effect on the onset or progression ofprion.