XIAP decreases caspase-12 cleavage and calpain activity in spinal cord of ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons. The cause for nerve cell demise is not clear but involves activation of the caspase family of cysteine proteases. We have shown that ER stress and caspase-12 activation occur in ALS transgenic mice carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. In these mice, we found that the antiapoptotic proteins, X-linked Inhibitor of Apoptosis Protein (XIAP) and the related protein, MIAP2 were decreased. To study the role of this, we generated double transgenic mice expressing XIAP in ALS spinal cord neurons using the Thy1 promoter. Overexpression of XIAP inhibited caspase-12 cleavage and reduced calpain activity in the ALS mice. XIAP also reduced the breakdown of calpastatin that is an inhibitor of calpain. In the double transgenic mice, life span was increased by about 12%. These data support the view that XIAP has beneficial effects in ALS and extends survival. The neuroprotective effect of XIAP involves inhibition of caspases and the stabilization of the calpastatin/calpain system that is altered in the ALS mice.     

利用转基因小鼠及转染色体小鼠产生人抗体的研究进展

自单克隆抗体（mAb)技术问世以来,解决了生命科学的许多重要问题,但其鼠源生导致的HAMA反应在大大限制了它在人体治疗中的应用,因此,制备人抗体成了亟待解决的难题,转入Ig基因组小鼠与转梁色体小鼠 的构建成功为解决这一难题提供了可行途径,本文就这两条小鼠的构建及其在制备人抗体中的应用进展作一综述。     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

Spatial learning deficit in transgenic mice that conditionally over-express GSK-3beta in the brain but do not form tau filaments

Deregulation of glycogen synthase kinase-3 (GSK-3) activity in neurones has been postulated as a key feature in Alzheimer's disease (AD) pathogenesis. This was further supported by our recent characterization of transgenic mice that conditionally over-express GSK-3beta in hippocampal and cortical neurones. These mice, designated Tet/GSK-3beta, showed many of the biochemical and cellular aspects of AD neuropathology such as tau hyperphosphorylation and somatodendritic localization, decreased nuclear beta-catenin, neuronal death and reactive gliosis. Tet/GSK-3beta mice, however, did not show tau filament formation up to the latest tested age of 3 months at least. Here we report spatial learning deficits of Tet/GSK-3beta mice in the Morris water maze. In parallel, we also measured the increase in GSK-3 activity while further exploring the possibility of tau filament formation in aged mice. We found a significant increase in GSK-3 activity in the hippocampus of Tet/GSK-3beta mice whereas no tau fibrils could be found even in very old mice. These data reinforce the hypothesis of GSK-3 deregulation in AD pathogenesis, and suggest that Tet/GSK-3beta mice can be used as an AD model and, most remarkably, can be used to test the therapeutic potential of the selective GSK-3 inhibitors that are currently under development. Additionally, these experiments suggest that destabilization of microtubules and alteration of intracellular metabolic pathways contribute to AD pathogenesis independent of toxicity triggered by the aberrant tau deposits.     

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants

Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the -glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.     

Regulation of insulin-like growth factor-l and binding protein-3 expression in oMtla-oGH transgenic mice

Growth hormone (GH)-transgenic mice provide a model for studying hormonal regulation of gene products responsible for efficient lean growth. Insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (BP-3) are two products involved in mediating the growth promoting actions of GH. Mice carrying the ovine metallothionein la-ovine growth hormone (oMtla-oGH) transgene were used to study GH regulation of IGF-I and PB-3 expression because these mice do not exhibit elevated basal oGH levels without transgene stimulation by exogenous zinc. C57B1/6XCBA mice with (transgenic=TG) and without (control=C) the oMtla-oGH transgene were activated (+Zn) or inactivated (-Zn) by the addition or removal of 25 mM zinc sulfate in the drinking water. Plasma IGF-I and BP-3 levels were determined by radioimmunoassay and western ligand blotting, respectively. Hepatic IGF-I and BP-3 mRNA levels were determined by slot-blot analysis. TG+Zn mice had higher plasma IGF-I (p<0.05) and hepatic IGF-I mRNA (p<0.05) levels as compared to TG-Zn, C+Zn and C-Zn mice. Plasma IGF-I and hepatic IGF-I mRNA levels in TG-Zn mice were not different from C+Zn and C-Zn mice. Removal of Zn decreased hepatic IGF-I mRNA levels to C levels in TG mice. Plasma BP-3 and hepatic BP-3 mRNA levels in TG+Zn mice were increased (p<0.05) as compared to TG-Zn, C-Zn and C+Zn. Plasma BP-3 and hepatic BP-3 mRNA levels did not differ between TG-Zn, C-Zn and C+Zn mice. Expression of the transgene also increased the level of plasma BP-3 during pregnancy as compared to that observed for pregnant C mice. We conclude that oGH regulates IGF-I and BP-3 expression in the oMtla-oGH transgenic mouse model system.     

Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum

Conventional screening protocols for transgene integration in mice employ tail tips or blood samples as sources to obtain genomic DNA preparations. We have developed a simple alternative non-surgical method. Epithelial cells are scraped off the inner surface of the rectum with a sterile plastic inoculation loop and are lysed with Kawasaki buffer. The lysate can be directly examined in a polymerase chain reaction (PCR) analysis without any need for further DNA purification. This procedure causes minimal harm and stress to the animals and repeated samples can be obtained as often as necessary. This technique has been used successfully to identify transgenic mice from a number of different lines. The method allows quick screening of numerous animals and contributes to a reduction of the number of surgical biopsies required     

Isolation and Identification of Novel ADP-Ribosylated Proteins from Streptomyces coelicolor A3(2)

An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.     

Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice

In the dentate gyrus of the mouse hippocampus, presynaptic recruitment of norepinephrine in response to repeated-burst stimulation can be described in terms of an interaction between storage and readily releasable pools. The dynamics of this interaction depends on neuronal activity (bursting), so that the higher the demand for norepinephrine, the faster it is delivered from the storage pool. We also found that alpha-synuclein, a presynaptic protein that plays a crucial role in dopamine compartmentalization in the striatum, is also involved in the compartmentalization of norepinephrine in the dentate gyrus. Experiments in transgenic mice with modified or absent alpha-synuclein revealed that the familial Parkinson's disease-linked alpha-synuclein mutation A30P can cause selective changes in the function of noradrenergic terminals. Addition of mutated human alpha-synuclein abolished the normal norepinephrine mobilization. There were no compensatory mechanisms available in the norepinephrine presynaptic terminals. In contrast, deletion of mouse alpha-synuclein is compensated for by increased vesicle transport from the storage pool. The effects are essentially the same as previously reported for dopaminergic terminals in the striatum, indicating that the important role of alpha-synuclein in neurotransmitter mobilization is not limited to dopaminergic terminals.     

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-aceB gene-ovine growth hormone (GH) gene (3 GH sequence) construct was fused to the ovine MT-Ia promoter-aceA gene-ovine GH gene (3 GH sequence). Therefore, in this single DNA sequence, bothaceA andaceB are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of theaceB-aceA gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressedEscherichia coli cells.     

ALS转基因小鼠脊髓中miRNA-132表达减少BDNF表达增加

目的研究miRNA-132和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)在肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)转基因小鼠脊髓中的表达变化,探讨miRNA-132、BDNF在ALS发病中的作用。方法取SOD1-G93A ALS转基因鼠发病早期(95d)、中期(108d)和晚期(122d)脊髓组织,应用qRT-PCR及原位杂交(hybridization in situ, ISH)技术检测miRNA-132的表达及定位,应用qRT-PCR及Western blot技术检测BDNF在mRNA及蛋白水平变化,免疫荧光技术检测BDNF在脊髓中的表达及分布,以同窝野生型鼠作为对照。结果与野生型鼠比较,miRNA-132在ALS转基因鼠脊髓组织表达下降,miRNA-132阳性信号主要定位脊髓前角细胞胞体;在ALS转基因鼠脊髓组织BDNF mRNA及蛋白水平均增高,BDNF免疫阳性细胞主要表达于脊髓前角神经元,表达信号明显增强。结论 miRNA-132、BDNF可能在ALS发病过程中发挥了重要作用。     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling

Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways.     

Cloning and sequencing of the CRABP-I locus from chicken and pufferfish: analysis of the promoter regions in transgenic mice

Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo