Gaëlle Tilly

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy.

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrPres) isolated from brains of affected animals or humans. However, alternate PrPres signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrPres of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.

Efficient and specific down-regulation of prion protein expression by RNAi

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.

Goat SRY induces testis development in XX transgenic mice

The testis‐determining gene SRY is not well‐conserved among mammals, particularly between mouse and other mammals, both in terms of protein structure and of expression regulation. To evaluate SRY phylogenic conservation in regards to its function, we expressed the goat gene (gSRY) in XX transgenic mouse gonads. Here, we show that gSRY induces testis formation, despite a goat expression profile. Our results demonstrate that sex‐reversal can be induced in XX‐mice by a non‐mouse SRY thus suggesting a conserved molecular mechanism of action of this testis‐determining gene across mammalian species.

Goat PRND expression pattern suggests its involvement in early sex differentiation

Expression of the goat prion protein gene locus was assessed by reverse transcriptase‐polymerase chain reaction on testes and ovaries at various developmental stages. A weak and stochastic expression of the PRNP and PRNT genes was observed. For PRNT, it is consistent with the detected deletions of two single nucleotides within its open reading frame in ruminant genes. PRND was expressed in both tissues at all stages. Whereas its expression is constant in the ovaries, it increases in testes between 36 and 46 days postcoitum (dpc) and remains high thereafter. In testes, Doppel was found in the nucleus of germinal cells and in the cytoplasm of Leydig cells at 44 dpc. It was detected in the cytoplasm of Leydig cells and of some Sertoli and germinal cells at 62 dpc. In the ovaries, it was observed in the nucleus of germinal cells at 44 dpc and mainly in their cytoplasm at 62 dpc. This expression pattern was shown to parallel that of C‐kit and suggests Doppel involvement in early testis differentiation. Developmental Dynamics 236:836–842, 2007.

Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

BackgroundThe physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages.ResultsOnly subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences.ConclusionsThese results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.

Divergent prion strain evolution driven by PrP C expression level in transgenic mice

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice : Application to Prnp

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet‐operator sequences inserted 5′ to exon 1 and a mouse neurofilament genomic clone carrying the chimeric‐repressor TRSID cDNA. In bi‐transgenic mice, ovine PrPC expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID‐silencer to precisely control temporal and spatial gene expression in vivo.

Unexpected high testis‐specific transcriptional activity of the cyclin T1 promoter in transgenic mice

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV‐1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5′‐flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell‐specific expression of the prion protein. These data re‐question the suspected location of the cyclin T1 gene regulatory elements.