Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

A cell-based assay system for activators of the environmental cell stress response.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Folate deficiency enhances the inflammatory response of macrophages.

B-vitamin deficiency is a risk factor for vascular disease. The mechanism by which the deficiency impacts on disease risk is unclear. We have analysed whether the inflammatory response of mononuclear cells can be modified by cellular folate status in vitro. We show that the mouse monocyte cell line RAW264.7 grown under folate restriction displays a decrease in intracellular folate levels and a reduced growth rate. The cells also show a 2- to 3-fold increase in expression of the inflammatory mediators, IL1ß, IL6, TNFα and MCP1 at the RNA and protein level (p<0.01) under conditions of folate deficiency. In contrast the production of the vaso-protective mediator nitric oxide is significantly reduced under these conditions. These metabolic changes are independent of the concentration of homocysteine in the medium and occur in the absence of significant changes in global DNA methylation. Folate deficiency may therefore exacerbate cardiovascular disease by augmenting pro-inflammatory signals in the monocyte-macrophage lineage.

Milk lacking α-casein leads to permanent reduction in body size in mice.

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

Eomesodermin is expressed in mouse oocytes and pre-implantation embryos.

T-box genes are a highly conserved family of genes encoding transcription factors, which share a conserved DNA binding domain (the T-box). Appropriate temporal and spatial expression of this gene family is critical for gastrulation and organogenesis in a number of species. The T-box containing gene Eomesodermin was first identified in Xenopus, where it plays a critical role in mesoderm formation. In situ analyses in mice have described the expression patterns of the mouse ortholog of this gene mEomesodermin (mEomes) at the time of implantation and during fetal development. Additional studies involving the disruption of the mEomes gene, have demonstrated an additional role for mEomes in trophoblast formation. However, these analyses did not address the possibility that maternally encoded or pre-blastocyst zygotic transcription of mEomes may also contribute to embryonic development. We show here that mEomes mRNA is present prior to blastocyst formation, and that the protein product of mEomes is associated with nuclear DNA during oocyte development and persistently localizes within all nuclei of the preimplantation embryo until the early blastocyst stage. mEomes protein is associated with the meiotic spindle in the unfertilized egg and with the mitotic spindle at each cell division. Our results are consistent with mEomesodermin having a role in early preimplantation development and inner cell mass formation in addition to its function in the trophoblast lineage.

Impaired glucose homeostasis and mitochondrial abnormalities in offspring of rats fed a fat-rich diet in pregnancy.

We previously reported that prenatal and suckling exposure to a maternal diet rich in animal fat leads to cardiovascular dysfunction in young adult rat offspring with subsequent development of dyslipidemia and hyperglycemia. We have further investigated glucose homeostasis in adult female offspring by euglycemic-hyperinsulinemic clamp and by dynamic assessment of glucose-stimulated insulin secretion in isolated, perifused pancreatic islet cells. Additionally, given the link between reduced mitochondrial DNA (mtDNA) content and the development of type 2 diabetes mellitus, we have measured mtDNA in organs from young adult animals. Sprague-Dawley rats were fed a diet rich in animal fat or normal chow throughout pregnancy and weaning. Infusion of insulin (5 mU.kg(-1).min(-1)) resulted in a higher steady-state plasma insulin concentration in 1-year-old offspring of fat-fed dams (OHF, n = 4) vs. offspring of control dams (OC, n = 4, P < 0.01). Glucose-stimulated insulin secretion in isolated islets from 9-mo-old OHF was significantly reduced compared with OC (n = 4, P < 0.05). Transmission electron micrography showed altered insulin secretory granule morphology in OHF pancreatic beta-cells. Kidney mtDNA was reduced in 3-mo-old OHF [16S-to-18S gene ratio: OC (n = 10) 1.05 +/- 0.19 vs. OHF (n = 10) 0.66 +/- 0.06, P < 0.05]. At 6 mo, gene chip microarray of OHF aorta showed reduced expression of the mitochondrial genome. Prenatal and suckling exposure to a diet rich in animal fat leads to whole body insulin resistance and pancreatic beta-cell dysfunction in adulthood, which is preceded by reduced tissue mtDNA content and altered mitochondrial gene expression.

Mitochondrial DNA turnover occurs during preimplantation development and can be modulated by environmental factors.

There is increasing evidence in humans that abnormal mitochondrial DNA (mtDNA) is associated with common degenerative disorders of the twenty-first century. MtDNA is exclusively female in origin and abnormalities in mtDNA can either be inherited, or generated de novo by adverse environmental factors that disturb mitochondrial DNA synthesis or destabilize mtDNA. The preimplantation period of development in mammals was thought to be relatively immune from environmentally induced changes to mtDNA, since no replication of mtDNA was thought to occur at this stage. This study demonstrates that there is a very short period of mtDNA synthesis immediately after fertilization, which can be affected by environmental stress. Adverse culture conditions during this phase of development could therefore alter the mitochondrial genome, with possible long-term consequences for the health of the offspring. The findings have relevance for all assisted reproduction programmes and for the rapidly emerging field of stem cell technologies.

Serum concentrations of homocysteine are elevated during early pregnancy in rodent models of fetal programming.

Maternal malnutrition can lead to fetal abnormalities and increase susceptibility to disease in later life. Rat models have been developed to study the physiology and metabolism underlying this phenomenon. One particular model of 50 % protein restriction during pregnancy, the low-protein diet (LPD) supplemented with methionine, has been developed to investigate the underlying mechanisms. Recent studies have shown that rats fed a LPD during only the first 4 d of pregnancy produce offspring that develop hypertension. These results suggest that the very earliest stages of embryo development are susceptible to diet-induced heritable changes. We demonstrate a marked elevation of maternal serum homocysteine (hcy) concentrations during the initial phases of pregnancy in both rats and mice fed an LPD. Fetal growth and many of the circulating amino acids are similarly perturbed in both rats and mice fed the LPD during pregnancy, indicating that the response to the LPD diet is similar in rats and mice. These findings allow us to exploit the advantages of the mouse experimental system in future analyses aimed at understanding the molecular basis of fetal programming. Our present findings are discussed with particular reference to mechanisms which may initiate fetal programming, and to the feasibility of dietary interventions aimed at reducing early pregnancy loss and pre-eclampsia in man.