Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis

The mannose-binding lectin (MBL) (also known as the mannose-binding protein) is a serum protein that plays a role as an "ante-antibody" in innate immunity. In man, MBL is encoded by a single gene, whereas in mice there are two homologous proteins, MBL-A and MBL-C. In order to evaluate the relative roles of these two forms of MBL, we created MBL-A null mice that were MBL-C sufficient. We found MBL-A null mice had enhanced survival in a septic peritonitis model compared to wild-type mice and complement 3 null mice at 24 h, 48 h and 10 d (P < 0.05). Reconstitution of these mice with human MBL reversed the phenotype. Surviving mice had significantly decreased TNF-alpha and IL-6 levels in the blood and peritoneal cavity (P < 0.01). In vitro studies indicate that bacteria opsonized with MBL-A-deficient serum induced significantly less cytokine by peritoneal macrophages compared to those with wild-type serum. Our results indicate that MBL-A is a modulator of inflammation in vivo and in vitro in the mouse and that the role of MBL may extend beyond its role as an opsonin.     

PTEN augments staurosporine-induced apoptosis in PTEN-null Ishikawa cells by downregulating PI3K/Akt signaling pathway

Staurosporine is a potent apoptosis inducer, but its mechanism remains to be clarified. We investigated the involvement of PTEN in staurosporine-induced apoptosis. Ishikawa cells, from an endometrial carcinoma cell line, expressed a high amount of PTEN mRNA but did not express the PTEN protein because of protein truncations. We isolated clones expressing the steady-state level of the PTEN protein from PTEN-null Ishikawa cells by transfection. The obtained clones showed reduced proliferative activity and reduced anchorage-independent cell growth with the augmented p27(Kip1). These cell lines were sensitized to apoptosis by staurosporine. A low concentration of UCN-01 did not affect apoptosis, but a high concentration augmented apoptosis in the PTEN-expressing clone. Alpha-sphingosine and H-7 did not affect apoptosis in these cell lines. PI3K inhibition augmented staurosporine-induced apoptosis in the parental cell line, but not in the PTEN-expressing clone. In the clone, phosho-Akt/PKB and phospho-Bad (Ser-136) were downregulated. Staurosporine reduced the levels of phospho-Akt/PKB and phospho-Bad (Ser-136) in all the cell lines, but the reduction was most significant in the PTEN-expressing clone. These results suggest that inhibition of the PI3K/Akt/PKB signaling pathway might be associated with staurosporine-induced apoptosis in Ishikawa cells.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Cardiac gene expression profile and lipid accumulation in response to starvation

Starvation induces many biochemical and histological changes in the heart; however, the molecular events underlying these changes have not been fully elucidated. To explore the molecular response of the heart to starvation, microarray analysis was performed together with biochemical and histological investigations. Serum free fatty acids increased twofold in both 16- and 48-h-fasted mice, and cardiac triglyceride content increased threefold and sixfold in 16- and 48-h-fasted mice, respectively. Electron microscopy showed numerous lipid droplets in hearts of 48-h-fasted mice, whereas fewer numbers of droplets were seen in hearts from 16-h-fasted mice. Expression of 11,000 cardiac genes was screened by microarrays. More than 50 and 150 known genes were detected by differential expression analysis after 16- and 48-h-fasts, respectively. Genes for fatty acid oxidation and gluconeogenesis were increased, and genes for glycolysis were decreased. Many other genes for metabolism, signaling/cell cycle, cytoskeleton, and tissue antigens were affected by fasting. These data provide a broad perspective of the molecular events occurring physiologically in the heart in response to starvation.     

The lesion of the rat substantia nigra pars compacta dopaminergic neurons as a model for Parkinson's disease memory disabilities

1. In this article we review the studies of memory disabilities in a rat model o Parkinson's disease (PD).2. Intranigral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to rats causes a partial lesion in the substantia nigra, compact part (SNc) and a specific loss of dopamine and its metabolites in the striatum of rats.3. These animals present learning and memory deficits but no sensorimotor impairments, thus modeling the early phase of PD when cognitive impairments are observed but the motor symptoms of the disease are barely present.4. The cognitive deficits observed in these animals affect memory tasks proposed to model habit learning (the cued version of the water maze task and the two-way active avoidance task) and working memory (a working memory version of the water maze), but spare long-term spatial memory (the spatial reference version of the Morris water maze).5. The treatment of these animals with levodopa in a dose that restores the striatal level of dopamine does not reverse these memory impairments, probably because this treatment promotes a high level of dopamine in extrastriatal brain regions, such as the prefrontal cortex and the hippocampus.6. On the other hand, the adenosine receptor antagonist, caffeine, partly reverse the memory impairment effect of SNc lesion in these rats. This effect may be due to caffeine action on nigrostriatal neurons, since it induces dopamine release and modulates the interaction between adenosine and dopamine receptor activity.7. These results suggest that the MPTP SNc-lesioned rats are a good model to study memory disabilities related to PD and that caffeine and other selective A(2A) adenosine receptor antagonists are promising drugs to treat this symptoms in PD patients.