David C. Wight

Adverse Effects of Chronic Endogenous Sympathetic Drive Induced by Cardiac Gsα Overexpression

To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 +/- 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF) was not different from that of control mice at baseline (LVFS, 40 +/- 3% versus 36 +/- 2%; LVEF, 78 +/- 3% versus 74 +/- 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 micrograms/kg per minute) infusion were higher than the values in control mice (LVFS, 68 +/- 4% versus 48 +/- 3%; LVEF, 96 +/- 1% versus 86 +/- 3%; P < .05). Norepinephrine (NE, 0.2 micrograms/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 +/- 4% versus 47 +/- 3%; LVEF, 93 +/- 2% versus 85 +/- 3%; P < .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 +/- 2% with ISO (0.02 micrograms/kg per minute) and by only 13 +/- 2% in three wild-type control mice (P < .01). NE (0.1 micrograms/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 +/ 8% versus 28 +/- 4%, P < .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 +/- 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 +/- 1.4% versus 1.2 +/- 0.3%, P < .05) and in the cross-sectional area of myocytes (298 +/- 29 versus 187 +/- 12 micron2, P < .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs-adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.

Reporter genes in transgenic mice

Althoughin vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detectedin situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to producein vivo models of cancer; they have been employed for the study ofin vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplanation experiments. The most commonly usedin situ reporter gene islacZ, which encodes a bacterial β-galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatalin vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights intoin vivo biological mechanisms. The development of newin vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.

Transgenic mice: a decade of progress in technology and research

In little more than a decade, the techniques developed for altering the genetic makeup of laboratory and livestock animals and plants have changed the landscape of biological research. It is now possible to introduce virtually any cloned gene into the germ line and study the expression pattern and effects of the introduced gene, or transgene. This has allowed the extension of in vitro and in vivo cell-culture studies into whole animal systems in which the introduced gene is subject to all normal regulatory processes from the onset of development. Although there have been reports of foreign gene expression resulting from direct injection of DNA in animals (e.g., Wolff et al., 1990; Zhu et al., 1993), transgenic animals are the primary model system for examining molecular genetic phenomena in vivo.

Pullaritheca longii gen. nov. and Kerryia mattenii gen. et sp. nov., lower carboniferous cupules with ovules of the Hydrasperma tenuis-type

Abstract New specimens of cupules assignable to Hydrasperma longii Matten et al. have been collected at Oxroad Bay in southern Scotland. Two of these bear large well-developed ovules that are tightly packed within the cupules and reveal the features of the relatively mature fructification. The attached ovules fall within the range of structural variation that encompasses both the isolated ovules that are the type specimens of H. tenuis and cupulate ovules described as H. tenuis from the uppermost Devonian of Ireland. However, the cupules from Scotland and Ireland have distinctly different morphologies. This demonstrates that H. tenuis ovules are produced by at least two different kinds of plants and reveals that ovule structure alone is not always a reliable taxonomic indicator among primitive gymnosperms. The genus Hydrasperma is restricted to isolated ovules because it can not be determined which, if either of the plants, represented by the currently-known cupulate morphologies, actually bore the type specimens of H. tenuis. Plants represented by the ovulate cupules from Scotland and Ireland are named Pullaritheca longii gen. nov. and Kerryia mattenii gen. et sp. nov. respectively.

Non adaptive change in early land plant evolution

Primary vascular architecture of members of the Paleozoic Aneurophytales (Progymnosper-mopsida) is described. This architecture is somewhat more complex but fundamentally similar (homologous) to that of members of the Trimerophytina, putative ancestors of aneurophytes. It is suggested that the presence of complex stelar morphology in aneurophytes was epiphenomenal, a passive result of changes in growth and development in a trimerophyte-like ancestor. Specifically, I suggest that the evolutionary transformation in primary vascular architecture from haplostele to ribbed protostele was a direct consequence of changes that affected the vertical spacing and degree of organization of lateral appendages in early vascular plants. This view is in sharp contrast to adaptationist explanations of change in stelar morphology expressed by other authors and provides an example of non-adaptive change in evolution.

The Human Growth Hormone Transgene: Expression in Hemizygous and Homozygous Mice

Abstract Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5–fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.

FLP-mediated site-specific recombination in microinjected murine zygotes.

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.