Geneviève Jolivet

Prolactin signal transduction to milk protein genes: carboxy-terminal part of the prolactin receptor and its tyrosine phosphorylation are not obligatory for JAK2 and STAT5 activation.

In this study, we have developed several Chinese Hamster ovary (CHO) cell clones stably expressing various deletion mutant forms of the rabbit prolactin receptor (rbPRL-R) to better define the domains of the receptor involved in JAK2 kinase interaction, STAT5 activation, and to assess the role of tyrosine phosphorylation of the PRL-R in signal transduction. We observed that the box 1 region of the receptor was critical for productive interaction with JAK2 and its tyrosine phosphorylation after PRL stimulation. However, this region appeared to require the presence of additional cytoplasmic domain region(s), such as box 2, to exert its complete effect. In addition, we found that a mutant form lacking the 141 C-terminal residues lost the capacity to be tyrosine phosphorylated in response to PRL but remained able to activate JAK2 kinase and STAT5 transcription factor, indicating that it contained the minimal sequence required for STAT5 activation. The absence of tyrosine phosphorylation of this C-terminal rbPRL-R mutant upon PRL stimulation indicated that the phosphorylation of the PRL-R normally occured in the last 141 animo acids (aa) containing three tyrosines and was not absolutely necessary for induction of these early events in PRL signal transduction. Transfectant cell lines expressing wild type (WT) PRL-R and this C-terminal mutant form were able to induce CAT activity upon PRL stimulation when transiently transfected with the ovine-beta-lactoglobulin promoter, containing STAT5 recognition sites, fused to the CAT reporter gene. The comparison between transcriptional activity of these two receptor forms leads to the conclusion that the C-terminal region of the rbPRL-R, containing the physiological sites for tyrosine phosphorylation, is probably responsible for an amplification of the PRL signal to milk protein genes.

Structure of the gene encoding rabbit β-casein

The entire rabbit beta-casein-encoding gene and 400 bp upstream were sequenced. Eight introns, located essentially at a position similar to the corresponding gene in other species, were found. Strong homology with several casein-encoding genes from rabbit and from other species was observed in the upstream region of the gene. Repeated sequences of unknown function were also located within introns.

Gene expression following transfection of fish cells

Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.

Worsening of Diet-Induced Atherosclerosis in a New Model of Transgenic Rabbit Expressing the Human Plasma Phospholipid Transfer Protein

Objective—Plasma phospholipid transfer protein (PLTP) is involved in intravascular lipoprotein metabolism. PLTP is known to act through 2 main mechanisms: by remodeling high-density lipoproteins (HDL) and by increasing apolipoprotein (apo) B-containing lipoproteins. The aim of this study was to generate a new model of human PLTP transgenic (HuPLTPTg) rabbit and to determine whether PLTP expression modulates atherosclerosis in this species that, unlike humans and mice, displays naturally very low PLTP activity. Methods and Results—In HuPLTPTg rabbits, the human PLTP cDNA was placed under the control of the human eF1-&agr; gene promoter, resulting in a widespread tissue expression pattern and in increased plasma PLTP. The HuPLTPTg rabbits showed a significant increase in the cholesterol content of the plasma apoB-containing lipoprotein fractions, with a more severe trait when animals were fed a cholesterol-rich diet. In contrast, HDL cholesterol level was not modified in HuPLTPTg rabbits. Formation of aortic fatty streaks was increased in hypercholesterolemic HuPLTPTg animals as compared with nontransgenic littermates. Conclusion—Human PLTP expression in HuPLTPTg rabbit worsens atherosclerosis as a result of increased levels of atherogenic apoB-containing lipoproteins but not of alterations in their antioxidative protection or in cholesterol content of plasma HDL.

A MGF/STAT5 binding site is necessary in the distal enhancer for high prolactin induction of transfected rabbit αs1-casein-CAT gene transcription

The rabbit αs1‐casein gene contains a distal prolactin‐dependent enhancer 3442‐3285 bp 5′ to the site of initiation of transcription. We have reported previously that four DNA/protein‐binding sites (F1–F4) are located within this distal enhancer. We now show that one of this binding site (the F4 site) binds in vitro a MGF/STAT5‐like factor. The functional importance of the F4 site was estimated by cotransfection of CHO cells with a chimeric gene containing or not the F4 sequence linked to the (−391/+1774)CAT gene and a plasmid encoding the rabbit mammary prolactin receptor. The F4 site is necessary for maximal response of the enhancer to prolactin. However, this site has to be associated to the F1–F3 fragment. It can be replaced by a genuine MGF/STAT5‐binding site. A mutational analysis indicates that F4 and F1 sites are simultaneously involved to confer a high prolactin sensitivity.

Regulation by the extracellular matrix (ECM) of prolactin-induced αs1-casein gene expression in rabbit primary mammary cells: Role of STAT5, C/EBP, and chromatin structure

The aim of the present study was to understand how the extracellular matrix (ECM) regulates at the gene level the prolactin (Prl)‐induced signal transducer and activator of transcription 5 (STAT5)‐dependent expression of the αs1‐casein gene in mammary epithelial cells. CCAAT enhancer binding proteins (C/EBPs) are assumed regulators of β‐casein gene expression. Rabbit primary mammary cells express αs1‐casein gene when cultured on collagen and not on plastic. Similar C/EBPβ, C/EBPδ, STAT5, and Prl‐activated STAT5 were found under all culture conditions. Thus the ECM does not act through C/EBPs or STAT5. This was confirmed by transfections of rabbit primary mammary cells by a construct sensitive to ovine prolactin (oPrl) and ECM (6i TK luc) encompassing STAT5 and C/EBP binding sites. The mutation of C/EBPs binding sites showed that these sites were not mandatory for Prl‐induced expression of the construct. Interestingly, chromatin immunoprecipitation by the anti‐acetylhistone H4 antibody (ChIP) showed that the ECM (and not Prl) maintained a high amount of histone H4 acetylation upstream of the αs1‐casein gene especially at the level of a distal Prl‐ and ECM‐ sensitive enhancer. Alpha6 integrin (a membrane receptor of laminin, the principal active component of the mammary ECM) was found at the surface of cells cultured on collagen but not on plastic. In cells cultured on collagen in the presence of anti‐α6 integrin antibody, Prl‐induced transcription of the endogenous αs1‐casein gene was significantly reduced, without modifying C/EBPs and STAT5. Besides, histone H4 acetylation was reduced. Thus, we propose that the ECM regulates rabbit αs1‐casein protein expression by local modification of chromatin structure, independently of STAT5 and C/EBPs. Copyright

Pig whey acidic protein gene is surrounded by two ubiquitously expressed genes

A 140-kb pig DNA fragment containing the whey acidic protein (WAP) gene cloned in a bacterial artificial chromosome (BAC344H5) has been shown to contain all of the cis-elements necessary for position-independent, copy-dependent and tissue-specific expression in transgenic mice. The insert from this BAC was sequenced. This revealed the presence of two other genes with quite different expression patterns in pig tissues and in transfected HC11 mouse mammary cells. The RAMP3 gene is located 15 kb upstream of the WAP gene in reverse orientation. The CPR2 gene is located 5 kb downstream of the WAP gene in the same orientation. The same locus organization was found in the human genome. The region between RAMP3 and CPR2 in the human genome contains a WAP gene-like sequence with several points of mutation which may account for the absence of WAP from human milk.

Structure of the gene encoding rabbit

The complete nucleotide sequence of the entire rabbit alpha s1-casein-encoding gene Aslca and its flanking regions was determined. These data represent the first complete primary sequence of an Aslca gene. The gene consists of 19 exons spread over 16 kb. Highly conserved sequences were found between this gene and other casein-encoding genes mainly upstream from the gene from position -180 to -10. Several repeated interspersed elements of unknown function were also identified within introns.

The insulator effect of the 5'HS4 region from the β-globin chicken locus on the rabbit WAP gene promoter activity in transgenic mice

Previous studies have shown that the 5′HS4 DNaseI hypersensitive site of the chicken β-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5′HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5′HS4 insulator (p5′HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5′HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5′HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5′HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5′HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.

Viral infection resistance conferred on mice by siRNA transgenesis

RNA interference is an attractive strategy to fight against viral diseases by targeting the mRNA of viral genes. Most studies have reported the transient delivery of small interfering RNA or small hairpin (shRNA) expression constructs. Here, we present the production of transgenic mice stably expressing shRNA or miRNA targeting the IE180 mRNA (immediate early gene) of the pseudorabies virus (PRV) which infects mice and farm animals. We firstly designed non-retroviral shRNA or miRNA expression vectors. Secondly, we selected the most efficient shRNA construct that targeted either the 5′part or 3′UTR of the IE mRNA and was able to knockdown the target gene expression in cultured cells, by measuring systematically the shRNA content and comparing this with the interfering effects. We then produced four lines of transgenic mice expressing different amounts of shRNA or miRNA in the brain but without signs of stimulation of innate immunity. Lastly, we tested their resistance to PRV infection. In all transgenic lines, we observed a significant resistance to viral challenge, the best being achieved with the shRNA construct targeting the 3′UTR of the IE gene. Viral DNA levels in the brains of infected mice were always lower in transgenic mice, even in animals that did not survive. Finally, this work reports an effective strategy to generate transgenic animals producing shRNA from non-retroviral expression vectors. Moreover, these mice are the first transgenic animal models producing shRNA with a significant antiviral effect but without any apparent shRNA toxicity.