Rapid genotyping of mice with hemoglobinopathies and globin transgenes

Because of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10(-18) M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3delta amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3delta, TNF alpha or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.     

Age-dependent modulation of circadian parameters in the field mouse Mus booduga

BACKGROUND AND PURPOSE: The levator palpebrae superioris (LPS) muscle courses anteriosuperiorly to culminate cranial to the posteriosuperior surface of the globe from where it courses anterioinferiorly to the trasal plate. Whitnall's superior transverse ligament (STL) has been suggested to suspend the LPS at its culmination. If this was the case, one would expect the STL to be located near the culmination of the LPS. In order to elucidate this functional aspect of the STL, the spatial relation of the STL of the LPS muscle is investigated in this study. METHODS: Surface coil MRI in an oblique sagittal plane along the optic nerve was performed in 6 orbits from 3 human cadavers in which the STL was marked with synthetic material. RESULTS: The MR images showed that in human cadaver specimens the STL is situated in the anterior descending portion of the LPS. CONCLUSION: This result suggests that the STL does not suspend the LPS at its culmination and is therefore not responsible for the curved course of the muscle.     

Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune thyroiditis

Four cases of ocular myopathy are presented. Their clinical, electromyographical and hystological signs are discussed comparatively with clinical forms described in the literature. One case was of pure ocular myopathy, without the affectation of other skeletal muscles. The other three cases were included in the descendent ocular myopathy, presenting a motor deficiency at the centure and inferior member levels. In all cases modifyings have been found in the EMG trajectory and the muscular biopsy of skeletal and/or ocular muscles.     

Shortening of the poly(A) region of mouse globin messenger RNA

Nucleated erythroid cells isolated from the spleens of anemic mice were used to investigate the processing of the polyadenylic acid region of globin mRNA. Cells were labeled in media containing [3H] adenosine and transferred to media containing no radioactive precursor and incubated further in the presence or absence of actinomycin D. After various times following the transfer of the cells, globin mRNA was isolated using a combination of oligo(dT)-cellulose affinity chromatography, sucrose density centrifugation, and globin cDNA (the complementary DNA copy of globin mRNA)-cellulose affinity chromatography. The size of the poly(A) region was determined by polyacrylamide gel electrophoresis of the T1 and pancreatic RNase-resistant fragments. The prelabeled poly(A) region which initially comprises approximately 150 adenylate residues was found to become shorter with time, both in cells incubated in medium containing no radioactive precursor and in the presence of actinomycin D. After 9 h of incubation in the presence of actinomycin D, two major size classes of poly(A) were observed, one containing 35 to 45 adenylic acid residues and the other containing 55 to 65 residues. These two size classes are similar to those found in circulating reticulocytes suggesting that the poly(A) shortening observed in these cell incubation studies is similar to that which occurs in vivo. Two protein synthesis inhibitors, emetine and cycloheximide, were investigated with respect to their effect on poly(A) shortening. Neither drug inhibited the shortening of the poly(A) region of globin mRNA, suggesting that protein synthesis is not required for this process to occur.     

Age-dependent penetrance of disease in a transgenic mouse model of familial amyotrophic lateral sclerosis

The mutation gly93-->ala of Cu,Zn superoxide dismutase (SOD) is found in patients with familial amyotrophic lateral sclerosis and causes motor neuron disease when expressed in transgenic mice. The progression of clinical and pathological disease was studied in a line of mice designated G1H. Clinical disease started at 91 +/- 14 days of age with fine shaking of the limbs, followed by paralysis and death by 136 +/- 7 days of age. Pathological changes begin by 37 days of age with vacuoles derived from swollen mitochondria accumulating in motor neurons. At the onset of clinical disease (90 days), significant death of somatic motor neurons innervating limb muscles has occurred; mice at end-stage disease (136 days) show up to 50% loss of cervical and lumbar motor neurons. However, neither thoracic nor cranial motor neurons show appreciable loss despite vacuolar changes. Autonomic motor neurons also are not affected. Mice that express wild-type human Cu,Zn SOD remain free of disease, indicating that mutations cause neuron loss by a gain-of-function. Thus, the age-dependent penetrance of motor neuron disease in this transgenic model is due to the gradual accumulation of pathological damage in select populations of cholinergic neurons.     

Regulation of human globin gene expression in mouse erythroleukemia x human fibroblast hybrid cells

A somatic cell hybrid, XX-8, was obtained from a fusion of tetraploid mouse erythroleukemia cells with human Lesch-Nyhan skin fibroblasts. This hybrid cell was previously shown (1) to produce human beta- but no human gamma-globin mRNA sequences after induction with dimethylsulfoxide. In this study we show that: (a) human beta- and gamma-globin genes are present in XX-8 cells in approximately equal numbers; (b) no human gamma-globin mRNA sequences can be detected in either the cytoplasmic or nuclear RNA fractions even with several different inducers; (c) after induction the human beta-globin gene is converted from a DNase I insensitive or closed structure to a DNase I open configuration, while the human gamma-globin gene remains closed; and (d) no human beta-globin polypeptide can be detected in the intact induced cells, indicating that fibroblast globin genes, even when induced to make mRNA in an erythroid environment, do not synthesize an RNA that is translated efficiently.     

Age-dependent phenotypic characteristics of a triple transgenic mouse model of Alzheimer disease.

The triple-transgenic mouse line (3×Tg-AD) harboring PS1M146V, APPSwe, and taup301L transgenes represents the only transgenic model for Alzheimer's disease (AD) to date capturing both β-amyloid and tau neuropathology. The present study provides an extensive behavioral characterization of the 3×Tg-AD mouse line, evaluating the emergence of noncognitive and cognitive AD-like symptoms at two ages corresponding to the early (6-7 months) and advanced (12-13 months) stages of AD-pathology. Enhanced responsiveness to aversive stimulation was detected in mutant mice at both ages: the 3×Tg-AD genotype enhanced acoustic startle response and facilitated performance in the cued-version of the water maze. These noncognitive phenotypes were accompanied by hyperactivity and reduced locomotor habituation in the open field at the older age. Signs of cognitive aberrations were also detected at both ages, but they were limited to associative learning. The present study suggests that this popular transgenic mouse model of AD has clear phenotypes beyond the cognitive domain, and their potential relationship to the cognitive phenotypes should be further explored. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.     

Conspiracy of silence among repeated transgenes

Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredictable from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of expression in vertebrate transgene arrays might result from interactions between repeats within the array. A recent experiment using transgenic mouse lines demonstrates that reduction in copy number of silenced transgenes within an array leads to a striking increase in expression, demonstrating that silencing is intrinsic to the array, and is not attributable to position effects of nearby sequences. This work calls into question functions that have been attributed to vertebrate locus control regions and boundaries, and draws attention to the notion that repeat-induced gene silencing is a system for protection of eukaryotic genomes against threatening sequence elements.     

Age-dependent variations in the sexual preference of male rats

Male rats were tested for their sexual preference behavior at either 37, 70, 90, or 150 days of age on three different occasions; while still sexually naive, after sexual experience with a receptive female, and while sexually aroused by having initiated copulation. These tests resulted in the following findings: a) 37-day-old sexually naive males showed a preference for other males and failed to show a preference for either sex after exposure to females; b) 70- and 90-day-old males showed a statistically significant preference for the female after acquiring sexual experience; c) 150-day-old animals showed a female-oriented preference only after being sexually aroused with two intromissions preceding the preference test, and d) none of the age groups tested showed a female-oriented preference without previous exposure to females. It was then concluded that a) female-oriented behavior requires sexual experience and b) the effects of experience varies with age.     

Age-dependent changes in the oxidative metabolism in rat brain

The effects of aging on the oxidation of labeled glucose and 3-hydroxybutyrate and on several mitochondrial enzymes in rat brain were investigated. The oxidation of labeled glucose and labeled 3-hydroxybutyrate was diminished by about 40 and 35%, respectively, in cerebral cortex slices from 2-year-old rats compared to those from 3-mo-old animals. A significant reduction in the activities of 3-hydroxybutyrate dehydrogenase, 3-oxo acid CoA transferase, acetoacetyl CoA thiolase, and NAD-isocitrate dehydrogenase was observed in brains of 1- and 2-year-old rats compared to 3-mo-old animals. However, aging had no effect on the activities of citrate synthase and pyruvate carboxylase. These findings show that specific alterations occur in the activities of several mitochondrial enzymes in aging brain.     

Mechanisms of developmental regulation in globin loci

The role of the c-myc oncogene has been little investigated in uveal melanoma. In this study an analysis of c-myc oncoprotein expression was undertaken using flow cytometry in 71 patients with posterior uveal melanoma. Nuclear c-myc oncoprotein was detected in all of the tumours, and survival analysis revealed a significant association between high oncoprotein positivity and improved survival (log rank test: chi2 = 6.47, P = 0.01). Multifactorial analysis using Cox's proportional hazards model revealed nuclear c-myc oncoprotein to be an independent prognostic marker more accurate than other clinicopathological parameters (log rank test: chi2 = 6.61, P = 0.01). However, this result of high oncoprotein expression correlating with improved outcome is surprising and in contrast to our previous studies using the same method on cutaneous melanoma, where high levels of nuclear c-myc expression have been found to correlate with poor outcome both in primary and secondary disease. This study suggests that the pattern of oncogene expression in uveal melanoma is distinct from cutaneous melanoma and that the underlying biology of these tumours is different.     

Expression and inheritance of multiple transgenes in rice plants

The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.     

The influence of chromosomal location on the expression of two transgenes in mice

We have generated mice having a single copy of the human haptoglobin gene (Hp2), driven by its natural promoter, and a neomycin resistance gene (Neo), driven by a herpes simplex thymidine kinase promoter with polyoma enhancers, inserted into two defined chromosomal locations, the Hprt locus on the X-chromosome and the apolipoprotein (apo) AI-CIII gene cluster on chromosome 9. The haptoglobin promoter is highly specialized in its tissue of action; the viral promoter has few restrictions. The apoAI-CIII gene is naturally active in only two tissues, whereas the Hprt gene region is ubiquitously active. Expression of both transgenes at substantial levels was achieved only (a) when the transgenes were inserted into the genome close to a known tissue-specific enhancer/locus control region in the apoAI-CIII gene cluster, and (b) when known conditions for function of their promoters were met. The specificities of the two chromosomal regions and of the two promoters are preserved, but their interactions are not specific. We conclude that transgenes are affected by locus-related enhancers in the same manner as nearby endogenous genes. Our experiments reinforce the usefulness of using gene targeting to direct single-copy transgenes to appropriate chromosomal locations.     

Effect of lead on globin synthesis in vitro

A defect in heme synthesis is well documented to occur in lead intoxication. Globin synthesis and the alpha/beta globin chain synthesis ratio have been shown to be disturbed in lead poisoning. To elucidate further the nature of the inhibitory effect of lead on hemoglobin synthesis, reticulocyte-rich peripheral blood samples have been incubated with lead at concentrations of 10(-6), 10(-5), and 10(-4) M, equivalent to 20, 200, and 2,000 mug/dl (.96, 9.6 and 96.0 mumol/l), respectively. The incorporation of tritiated leucine into globin was determined. Globin chain synthesis decreased to 85, 49 and 15% of the control value with increasing lead concentrations. The effect of heme on inhibition of globin synthesis by lead was studied by incubating reticulocyte-enriched blood with lead 10(-5) M (200 mug/dl; 9.6 mumol/l) in the presence or absence of heme at a concentration of 10(-3) M. Lead alone depressed the incorporation of tritiated leucine to 45% of control, while globin synthesis with heme alone was 109% of control. Although in the presence of both heme and lead, the mean incorporation of leucine into globin was 69.2% of control, the increase was not significantly different from that obtained with lead alone. It is concluded that (1) lead has an inhibitory effect on globin synthesis at a concentration considered to be within the acceptable range encountered in environmental lead pollution, and the extent of inhibition is dose-dependent; (2) the effect of lead on globin synthesis is not significantly prevented by heme and, therefore, does not appear to be mediated through the well-documented inhibitory effect of lead on heme synthesis.     

Age-dependent modifications in the metabolism of mevalonate pathway lipids in rat brain

Increased public and professional awareness has resulted in more women obtaining mammograms. As a result, the surgeon is often called on to diagnose and treat occult breast lesions. The development of new diagnostic modalities has changed the way such breast lesions are approached. Management decisions are made in the context of new pressures applied by the growing managed care imperative and increased mediocolegal exposure. In this review, we establish guidelines for the management of non-palpable breast abnormalities that place the welfare of the patient first.