A. John Clark

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long‐chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart‐type FABP (H‐FABP) exhibit a severe defect of peripheral (non‐hepatic, non‐fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and β‐hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H‐FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H‐FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.—Binas, B., Danneberg, H., McWhir, J., Mullins, L., Clark, A. J. Requirement for the heart‐type fatty acid binding protein in cardiac fatty acid utilization. FASEB J. 13, 805–812 (1999)

Stably Transfected Human Embryonic Stem Cell Clones Express OCT4‐Specific Green Fluorescent Protein and Maintain Self‐Renewal and Pluripotency

Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos; they can be cultured indefinitely and differentiated into many cell types in vitro. These cells therefore have the ability to provide insights into human disease and provide a potential unlimited supply of cells for cell‐based therapy. Little is known about the factors that are important for maintaining undifferentiated hESCs in vitro, however. As a tool to investigate these factors, transfected hES clonal cell lines were generated; these lines are able to express the enhanced green fluorescent protein (EGFP) reporter gene under control of the OCT4 promoter. OCT4 is an important marker of the undifferentiated state and a central regulator of pluripotency in ES cells. These OCT4‐EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells. Expression of OCT4‐EGFP is colocalized with endogenous OCT4, as well as the hESC surface antigens SSEA4 and Tra‐1‐60. In addition, the expression is retained in culture for an extensive period of time. Differentiation of these cells toward the neural lineage and targeted knockdown of endogenous OCT4 expression by RNA interference downregulated the EGFP expression in these cell lines, and this correlates closely with the reduction of endogenous OCT4 expression. Therefore, these cell lines provide an easy and noninvasive method to monitor expression of OCT4 in hESCs, and they will be invaluable for studying not only OCT4 function in hESC self‐renewal and differentiation but also the factors required for maintenance of undifferentiated hESCs in culture.

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV‐1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV‐1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.

Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain

To study the function of astrocytes in the adult brain, we have targeted the expression of E. coli nitroreductase (NTR) to the astrocytes of transgenic mice under the control of the GFAP promoter. The astrocytes expressing NTR were selectively ablated after administration of the prodrug CB1954, resulting in motor discoordination. Histological examination showed that the region most affected in the brain was the cerebellum, in which the Bergmann glia were eliminated and the granular neurons had degenerated. Specific effects were also noted on the dendrites of the Purkinje cells, and the junction between these neurons and granular layer was disrupted. Astrocyte ablation was associated with a dramatic decrease in the expression of glutamate transporters, which may account for the degeneration of granular neurons since the excitotoxic effects of glutamate result in a similar phenotype. These results provide the first evidence that astrocytes are important for the survival of neurons in the adult brain in vivo. GLIA 34:272–282, 2001.

Proliferative lifespan is conserved after nuclear transfer

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.

Gene targeting in primary fetal fibroblasts from sheep and pig.

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.

The mammary factor MPBF is a prolactin-induced transcriptional regulator which binds to STAT factor recognition sites

Site‐directed mutagenesis of the three binding sites for the mammary factor MPBF in the β‐lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon γ‐activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor‐induced transcription factors.

Telomerase-Immortalized Sheep Fibroblasts Can Be Reprogrammed by Nuclear Transfer to Undergo Early Development

Abstract Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50–200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.

A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland

BackgroundThis paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine β-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag).ResultsKIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells.ConclusionKIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland.

Characterization of the ovine LHβ-subunit gene: the promoter directs gonadotrope-specific expression in transgenic mice

The alpha- and beta-subunits of the gonadotropin hormones are expressed in the gonadotrope cells of the anterior pituitary. There are no adequate in vitro systems for the analysis of beta-subunit gene expression. In this study, therefore, transgenic mice have been used to investigate the regulation of expression of the ovine luteinizing hormone beta-gene (oLH beta) in vivo. oLH beta was isolated, characterized, and 1.9 kb of the promoter fused to the bacterial reporter chloramphenicol acetyl-transferase (CAT). Three lines of transgenic mice were generated. CAT enzyme was detected in the pituitary of two lines, whereas the third line did not express. Measurement of endogenous luteinizing hormone and follicle stimulating hormone levels in both expressing lines revealed small differences when compared to controls, but these did not affect the fertility of the animals. Immunostaining of the anterior pituitary revealed that the oLH beta CAT transgene was expressed specifically in gonadotrope cells.