Comparative analysis on the structural features of the 5' flanking region of κ-casein genes from six different species

κ-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of κ-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human κ-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the κ-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the κ-casein gene promoter

PLURABBIT - Analyse transcriptomique et caractérisation fonctionnelle de cellules souches pluripotentes de lapin pour la fabrication de nouveaux modèles animaux

National audienceLe projet PLURABBIT a pour objectif principal de développer de nouveaux outils moléculaires et cellulaires permettant d'explorer les mécanismes de la régulation de la pluripotence chez les mammifères. Les objectifs sépcifiques sont (1) la création de lignées de cellules souches pluripotentes chez le lapin, (ii) la caractérisation de leur transcriptome et (iii) l'analyse de leur capacité à contribuer au développement embryonnaire et foetal in vivo. Ce project ouvre des perspectives à moyen terme grâce à la possibilité de développer des applications biomédicales et pharmaceutiques dans le domaine des modèles animaux. Pour atteindre ces objectifs, le consortium réunit les expertises de quatre laboratoires académiques, deux français et deux hongrois, possédant tous une position leader au niveau européen dans le domaine des cellules souches et des biotechnologies chez le lapin. Le project s'appuie sur des découvertes récentes réalisées chez les rongeurs qui montrent qu'il est possible de créer in vitro deux types de cellules souches pluripotentes, l'une utilisant la signalisation LIF/STAT3 et l'autre la signalisation FGF2, pour se maintenir en autorenouvellement à l'état pluripotent indifférencié. Cette dichotomie entre les dépendances au LIF et au FGF2 s'applique aux cellules souches embryonnaires (ES) et aux cellules souches pluripotentes induites (iPS). Dans la première partie du projet, nous fabriquerons des lignées de cellules souches pluripotentes (ES et iPS) de lapin dépendantes du LIF et du FGF2, sur la base des protocoles pré-établis chez les rongeurs et les Primates. Dans la deuxième partie du projet, nous développerons de nouveaux outils (puces ADN et panel de miRNAs) que nous utiliserons afin de caractériser et comparer les cartes d'identité moléculaires de toutes les lignées produites. Dans la dernière partie du projet, nous évaluerons la capacité de ces lignées à coloniser l'embryon pré-implantatoire de lapin et ainsi contribuer au développement embryonnaire et foetal, y compris la lignée germinale. En parallèle, nous étudierons la capacité des cellules souches pluripotentes de lapin à être reprogrammées par transfert nucléaire dans l'ovocyte. Le projet PLURABBIT générera une importante quantité d'information bioinformatique qui servira à explorer la régulation de la pluripotence chez les espèces non murines. Il apportera également des informations cruciales sur la capacité des cellules souches pluripotentes de lapin à se développer en un organisme adulte et permettra de corréler cette capacité à des caractéristiques transcriptomiques données. Finalement, ce projet conduira au développement d'une technologie de transgénèse chez le lapin qui utilise les cellules souches pluripotentes

Special features of rabbit milk production

International audienc

Comparative analysis on the structural features of the 5' flanking region of <it>κ</it>-casein genes from six different species

Abstract κ-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of κ-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human κ-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the κ-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the κ-casein gene promoter.</p

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNARNARNA encoding the SB100X hyperactive transposase together with plasmid DNANA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNARNARNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ~2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNARNARNA encoding the SB100X hyperactive transposase, together with circular plasmid DNANA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNARNARNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ~3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors