Laurence Lepourry

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.

ABSTRACT The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrPVRQ-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrPVRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD.

Background The evolution of the variant Creutzfeldt-Jakob disease (vCJD) epidemic is hazardous to predict due to uncertainty in ascertaining the prevalence of infection and because the disease might remain asymptomatic or produce an alternate, sporadic-like phenotype. Methodology/Principal Findings Transgenic mice were produced that overexpress human prion protein with methionine at codon 129, the only allele found so far in vCJD-affected patients. These mice were infected with prions derived from variant and sporadic CJD (sCJD) cases by intracerebral or intraperitoneal route, and transmission efficiency and strain phenotype were analyzed in brain and spleen. We showed that i) the main features of vCJD infection in humans, including a prominent involvement of the lymphoid tissues compared to that in sCJD infection were faithfully reproduced in such mice; ii) transmission of vCJD agent by intracerebral route could lead to the propagation of either vCJD or sCJD-like prion in the brain, whereas vCJD prion was invariably propagated in the spleen, iii) after peripheral exposure, inefficient neuroinvasion was observed, resulting in an asymptomatic infection with life-long persistence of vCJD prion in the spleen at stable and elevated levels. Conclusion/Significance Our findings emphasize the possibility that human-to-human transmission of vCJD might produce alternative neuropathogical phenotypes and that lymphoid tissue examination of CJD cases classified as sporadic might reveal an infection by vCJD-type prions. They also provide evidence for the strong propensity of this agent to establish long-lasting, subclinical vCJD infection of lymphoreticular tissues, thus amplifying the risk for iatrogenic transmission.

High-level, stage- and mammary-tissue-specific expression of a caprine κ-casein-encoding minigene driven by a β-casein promoter in transgenic mice

Abstract A 5 truncated caprine (ca) κ-casein-encoding gene (κCas) was fused to the 3′ end of a 3′ truncated ca βCas. The βCas form comprised the 0.8-kb 3′ end of intron 2, the remaining part of the transcription unit containing codons -2 to stop 172, and 0.43 kb of the 3 flanking region. The βCas form comprised a 3-kb 5′ flanking region and the 5′ end of the transcription unit terminating 69 bp dowstream from exon 2 which encodes the 15-amino-acid (aa) signal peptide and the first 2 aa of mature βCas. The resulting hybrid gene driven by the βCas promoter was expressed in all eight lines of transgenic mice investigated, although at different levels. In two lines, the yield of recombinant (re-) κCas was ≥ 3 mg/ml of milk. The stage- and mammary tissue-specific expression was similar to that of endogenous βCas. The re-κCas differed from its goat milk counterpart by the occurrence of four extra aa at the N-terminal end, indicating that the signal peptidase released the βCas signal peptide. According to sedimentation analyses of murine milk containing ≥ 3 mg re-κCas/ml, the latter essentially occurred in micelles. Preliminary comparative assays of the behavior of αs1Cas-κCas and αs1Cas-reκCas mixtures upon incremental addition of Ca2+ showed that re-κCas had the capacity to protect αs1Cas against Ca2+-induced precipitation in forming stable micelles. In contrast, the expression of a reconstituted ca κCas gene was in the range of a few μg re-κCas/ml of milk in the five transgenic lines investigated.

Introduction of a Proximal Stat5 Site in the Murine α-Lactalbumin Promoter Induces Prolactin Dependency In Vitro and Improves Expression Frequency In Vivo

In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position −70 on a 560 bp murine α-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.

p28, a truncated form of TRα1 regulates mitochondrial physiology

We have previously identified in mitochondria two truncated forms of the T3 nuclear receptor TRα1, with molecular weights of 43kDa (p43) and 28kDa (p28) respectively located in the matrix and in the inner membrane. Previously, we have demonstrated that p43 stimulates mitochondrial transcription and protein synthesis in the presence of T3. Here we report that p28 is targeted into the organelle in a T3‐dependent manner and displays an affinity for T3 higher than the nuclear receptor. We tried to generate mice overexpressing p28 using the human α‐skeletal actin promoter, however we found an early embryonic lethality that was probably linked to a transient expression of p28 in trophoblast giant cells. This could be partly explained by the observation that overexpression of p28 in human fibroblasts induced alterations of mitochondrial physiology.

Distal element(s) is(are) required for position-independent expression of the goat α-lactalbumin gene in transgenic mice. Potential relationship with the location of the cyclin T1 locus

We recently reported the site-independent and copy-number-related expression in mice of a goat α-lactalbumin gene with 150 kb and 10 kb of 5- and 3-flanking sequences, respectively. In the present study, we observed that the resection of the 5-flanking region, leaving only 70 kb, resulted in a site-dependent expression of this milk protein-encoding transgene. This suggests that important cis-regulatory elements are located within the distal-deleted sequence. Within this region, we localised the promoter of the cyclin T1 gene, an ubiquitously expressed gene. So far, no other gene has been located between these two loci. Since these two genes are differentially expressed, our data suggest the potential location of an insulator within the deleted region that allows the two genes to be independently regulated.

Targeted Expression of the only Zinc Finger Gene in Transgenic Mice is Associated with Impaired Mammary Development

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat β-casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.