Solange Soulier

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.

Complete nucleotide sequence of bovine α-lactalbumin gene: comparison with its rat counterpart

Abstract The nucleotide sequence of the bovine α-lactalbumin gene, whose organization is very similar to that of its rat counterpart, was deduced from the analysis of 2 λ clones isolated from a HindIII genomic bank. The 3090 sequenced nucleotides comprise 738 bp upstream from the transcription unit (∼2 kb) which contains 4 exons of 160, 159, 76 and 330 bp separated by 3 introns of 321, 473 and 504 bp. Comparison with the rat α-lactalbumin gene shows similar percentages of homology between the 4 cognate exons. Since only the first three exons are homologous to the corresponding exons of the lysozyme gene, it is suggested that the 4th exons of α-lactalbumin and lysozyme genes have different origins. The bovine α-lactalbumin mRNA is 725 nucleotides long, excluding the poly(A) tail. The reading frame and the flanking 5′ and 3′ untranslated regions contain 429, 27 and 269 nucleotides, respectively. The derived amino acid sequence differs at 10 positions from that determined directly on mature α-lactalbumin.

Expression analysis of ruminant α-lactalbumin in transgenic mice: Developmental regulation and general location of important cis-regulatory elements

The bovine α‐lactalbumin transgene with 750 bp and 336 bp of the 5′ and 3′ flanking region, respectively, is developmentally regulated as its endogenous counterpart in transgenic mice. Comparative expression analysis of three 5′‐shortened constructs suggests that the region −4771/−220 contains important cis‐acting transcriptional elements. The level of expression of a long caprine α‐lactalbumin transgene encompassing 8.5 kb and 9.5 kb of the 5′ and 3′ flanking region, respectively, was higher but still unrelated to the copy number. Expression of the transgenes and of endogenous milk‐protein genes was tissue‐specific. In contrast with a recent report, only low amounts of the relevant mRNA were detected in some skin samples, which suggests a possible contamination by mammary tissue.

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives.

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.

The bovine α-lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice

A hybrid construct derived from ovine trophoblastin cDNA and bovine α‐lactalbumin‐encoding gene, was injected into the pronuclei of mouse eggs. In one of the resulting transgenic mouse lines, expression of the hybrid construct was detected and found to be limited to the mammary gland of lactating females which secreted active ovine trophoblastin. This strongly suggests that important cis‐acting DNA sequences involved in tissue‐specific expression of the bovine gene are located within the second half of the 3′ untranslated region, or/and the proximal 5′and 3′ regions flanking the transcriptional unit.

Complete nucleotide sequence of ovine α-lactalbumin mRNA

Abstract The nucleotide sequence of ovine α-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine α-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5′ non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature α-lactalbumin. The coding region is followed by a 3′ untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-α-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human α-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.

Sequence of the murine α-lactalbumin-encoding cDNA: interspecies comparison of the coding frame and deduced preprotein

Abstract The structure of the mouse α-lactalbumin-encoding mRNA was deduced from sequence analysis of eight cDNA clones. The almost full-length mRNA of 732 nucleotides [poly(A) tail excluded] and the deduced pre-protein share 85% and 86% homology with their rat counterpart, respectively. Interspecies comparison of the pre-protein showed the occurrence of an extra amino acid (aa) in the signal peptide and of two mutations affecting two reported invariant aa residues at positions 44 and 107, which weakens the assumption that both aa residues might play a significant structural and/or functional role.

The bovine and ovine genomes contain multiple sequences homologous to the α-lactalbumin-encoding gene

Bovine and ovine (pseudo)genes homologous to the alpha-lactalbumin-encoding gene are described. In both cases, sequence analysis reveals homology extending downstream from exon 2. Southern analysis indicates the presence of a family of alpha-lactalbumin-related sequences in the bovine genome.

Preparation and fractionation of goat κ-casein: analysis of the glycan and peptide components

Whole goat kappa-casein was prepared by chromatography of whole casein on hydroxyapatite. Chromatography of whole kappa-casein on DEAE-cellulose separated 5 fractions. All of them were sensitive to chymosin. Their amino acid and carbohydrate composition, phosphate content and molecular weight were determined. Galactose, N-acetylgalactosamine, N-acetyl and N-glycolyl neuraminic acids were identified in whole kappa-casein. It appears that goat kappa-casein, like cow, buffalo and ewe kappa-caseins, is composed of several fractions having identical peptide chains and differing in their carbohydrate contents. The main fraction, devoid of carbohydrate, was treated with chymosin. The para-kappa-casein and caseinomacropeptide were isolated. Their amino acid composition and phosphate content were determined.

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.