The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.     

Carbon monoxide induces murine thymocyte apoptosis by a free radical-mediated mechanism

Carbon monoxide (CO) induces acute or chronic toxicity, according to the level and duration of the exposure. Since chronic CO exposure was shown to have immunosuppressive effects (as it decreases the frequency of rat splenic immunocompetent cells and immunoglobulin production), we investigated the effect of CO on thymocytes, since these are the most sensitive cells to oxidative damage from the lymphoid lineage. We exposed thymocytes to CO, then determined their apoptotic index after 6 h of incubation at 37°C using the fluorochrome Hoechst 33342 and electron microscopy and found an increase of apoptosis in CO-exposed thymocytes. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an antioxidant vitamin E analog, decreased CO-induced thymocyte apoptosis unlike methylene blue, L-nitroarginine methyl ester or pyrrolidine dithiocarbamate. We also observed that lipid peroxidation was increased in the CO-exposed thymocytes and that it was inhibited by Trolox. Our results suggest that CO induces thymocyte apoptosis by a free radical-mediated mechanism which can be inhibited by Trolox but which does not involve the activation of the guanylyl cyclase–cGMP pathway.     

Kinetics of thymocyte developmental process in fetal and neonatal mice

Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4^hiCD8^hi (DP), TCR CD4^hiCD8^-/^loCD8^int/hi(CD4 SP), and TCR CD4^-/^loCD8^int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via im-mature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR^-CD4^-CD8^ cells, occurring between Fd 15 and Fd 17 and the other is via TCR^-CD4^ CD86-cells,occurring from Fd 17 until birth. In contrast, the TCR^-CD4^-CD8^ pathway dominated overwhelminglyin the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway pref-erence varies with mouse strains. With respect to the shift in the intensity of CD4 and CD8 expression onthymocytes from fetal to adult mice, the TCR CD4^hiCD8^-/^lo, and TCR^ CD4^-/^loCD8^int/hi subsets might be equivalent to the medullary type TCR^ CD4/CD8 SP cells.     

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.     

Enhanced viral resistance in transgenic mice expressing the human beta 1 interferon.

Recombinant plasmids pMTIF-beta 1A and pMTIF-beta 1B were constructed by fusing the metallothionein I promoter-regulatory region to the human beta 1 interferon (HuIFN-beta 1) gene. These linearized fusion genes were then introduced into mouse germ lines by zygote microinjection. The chromosomal integration and the germ line transmission of the injected DNA sequences in the resulting transgenic mice were detected by DNA dot blot and Southern transfer hybridizations. The sera of at least two strains of metallothionein/HuIFN transgenic mice were found to protect human WISH cells against vesicular stomatitis virus infection, and this activity could be neutralized by preincubation with anti-HuIFN-beta 1 antibody. These transgenic mice demonstrated significantly enhanced resistance to pseudorabies virus compared with nontransgenic mice when inoculated with pseudorabies virus. The level of resistance seemed to correlate with the concentrations of HuIFN-beta 1 in serum. These transgenic mice may be used as models to study IFN-induced responses and may serve as prototypes to generate disease-resistant animals.     

Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein

We have generated embryonic stem (ES) cells and transgenic mice carrying a tau-tagged green fluorescent protein (GFP) transgene under the control of a powerful promoter active in all cell types including those of the central nervous system. GFP requires no substrate and can be detected in fixed or living cells so is an attractive genetic marker. Tau-tagged GFP labels subcellular structures, including axons and the mitotic machinery, by binding the GFP to microtubules. This allows cell morphology to be visualized in exquisite detail. We test the application of cells derived from these mice in several types of cell-mixing experiments and demonstrate that the morphology of tau-GFP-expressing cells can be readily visualized after they have integrated into unlabeled host cells or tissues. We anticipate that these ES cells and transgenic mice will prove a novel and powerful tool for a wide variety of applications including the development of neural transplantation technologies in animal models and fundamental research into axon pathfinding mechanisms. A major advantage of the tau-GFP label is that it can be detected in living cells and labeled cells and their processes can be identified and subjected to a variety of manipulations such as electrophysiological cell recording.     

Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors

Genetically-encoded biosensors based on the principle of F?rster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.     

Dilated cardiomyopathy in transgenic mice expressing a mutant A subunit of protein phosphatase 2A

The protein phosphatase 2A (PP2A) holoenzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B. The PP2A core enzyme is composed of subunits A and C. Both the holoenzyme and the core enzyme are similarly abundant in heart tissue. Transgenic mice were generated expressing high levels of a dominant negative mutant of the A subunit (A delta 5) in the heart, skeletal muscle, and smooth muscle that competes with the endogenous A subunit for binding the C subunit but does not bind B subunits. We found that the ratio of core enzyme to holoenzyme was increased in A delta 5-expressing hearts. Importantly, already at day 1 after birth, A delta 5-transgenic mice had an increased heart weight-to-body weight ratio that persisted throughout life. Echocardiographic analysis of A delta 5-transgenic hearts revealed increased end-diastolic and end-systolic dimensions and decreased fractional shortening. In addition, the thickness of the septum and of the left ventricular posterior wall was significantly reduced. On the basis of these findings, we consider the heart phenotype of A delta 5-transgenic mice to be a form of dilated cardiomyopathy that frequently leads to premature death.     

Experimental allergic encephalomyelitis is inhibited in transgenic mice expressing human C-reactive protein

We show here using a transgenic model that human C-reactive protein (CRP) protects against experimental allergic encephalomyelitis (EAE) in C57BL/6 mice. In transgenic compared with wild-type females, the duration of the human CRP acute phase response that accompanies the inductive phase of active EAE correlates with a delay in disease onset. In transgenic males, which have higher human CRP expression than females do, EAE is delayed, and its severity is reduced relative to same-sex controls. Furthermore, in male transgenics, there is little or no infiltration of the spinal cord by CD3(+) T cells and CD11b(+) monocytes and macrophages, and EAE is sometimes prevented altogether. CRP transgenics also resist EAE induced passively by transfer of encephalitogenic T cells from wild-type donors. Human CRP has three effects on cultured encephalitogenic cells that could contribute to the protective effect observed in vivo: 1) CRP inhibits encephalitogenic peptide-induced proliferation of T cells; 2) CRP inhibits production of inflammatory cytokines (TNF-alpha, IFN-gamma) and chemokines (macrophage-inflammatory protein-1alpha, RANTES, monocyte chemoattractant protein-1); and 3) CRP increases IL-10 production. All three of these actions are realized in vitro only in the presence of high concentrations of human CRP. The combined data suggest that during the acute phase of inflammation accompanying EAE, the high level of circulating human CRP that is achieved in CRP-transgenic mice inhibits the damaging action of inflammatory cells and/or T cells that otherwise support onset and development of EAE.     

Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105-125

To identify sequence domains important for the neurotoxic and neuroprotective activities of the prion protein (PrP), we have engineered transgenic mice that express a form of murine PrP deleted for a conserved block of 21 amino acids (residues 105-125) in the unstructured, N-terminal tail of the protein. These mice spontaneously developed a severe neurodegenerative illness that was lethal within 1 week of birth in the absence of endogenous PrP. This phenotype was reversed in a dose-dependent fashion by coexpression of wild-type PrP, with five-fold overexpression delaying death beyond 1 year. The phenotype of Tg(PrPDelta105-125) mice is reminiscent of, but much more severe than, those described in mice that express PrP harboring larger deletions of the N-terminus, and in mice that ectopically express Doppel, a PrP paralog, in the CNS. The dramatically increased toxicity of PrPDelta105-125 is most consistent with a model in which this protein has greatly enhanced affinity for a hypothetical receptor that serves to transduce the toxic signal. We speculate that altered binding interactions involving the 105-125 region of PrP may also play a role in generating neurotoxic signals during prion infection.     

Enhanced virus resistance of transgenic mice expressing the human MxA protein.

MxA is a GTPase that accumulates to high levels in the cytoplasm of interferon-treated human cells. Expression of MxA cDNA confers to transfected cell lines a high degree of resistance against several RNA viruses, including influenza, measles, vesicular stomatitis, and Thogoto viruses. We have now generated transgenic mice that express MxA cDNA in the brain and other organs under the control of a constitutive promoter. Embryonic fibroblasts derived from the transgenic mice were nonpermissive for Thogoto virus and showed reduced susceptibility for influenza A and vesicular stomatitis viruses. The transgenic animals survived challenges with high doses of Thogoto virus by the intracerebral or intraperitoneal route. Furthermore, the transgenic mice were more resistant than their nontransgenic littermates to intracerebral infections with influenza A and vesicular stomatitis viruses. These results demonstrate that MxA is a powerful antiviral agent in vivo, indicating that it may protect humans from the deleterious effects of infections with certain viral pathogens.     

Differential regulation of cholesterol homeostasis in transgenic mice expressing human cholesterol ester transfer protein

We investigated whether expression of cholesterol ester transfer protein (CETP) in mice alters the regulation of cholesterol metabolism. Transgenic mice expressing human CETP (CETP-TG) and nontransgenic littermates (non-TG) were fed either a monounsaturated fatty acid (MUFA) or a saturated fatty acid (SFA)-rich diet in the presence or absence of cholesterol. Mice fed with MUFA diet had higher CETP activity compared with SFA-fed mice. Addition of cholesterol to the MUFA diet decreased CETP activity, whereas addition of cholesterol to the SFA diet had no effect. Cholesterol 7alpha-hydroxylase (Cyp7a) activity was higher in CETP-TG mice compared with non-TG mice when fed a MUFA diet, whereas SFA fed CETP-TG mice showed lower Cyp7a activity as compared with non-TG. Microsomal triglyceride transfer protein (MTTP) activity was higher in CETP-TG mice compared with non-TG mice when fed a MUFA diet. HMG-CoA reductase activity was lower in CETP-TG mice compared with non-TG mice when fed a MUFA or a SFA diet. These data demonstrate that the regulation of Cyp7a, HMG-CoA reductase, and MTTP is altered in CETP-TG mice as compared with non-TG mice and these alterations are further modulated by the quality of dietary fats. These findings highlight the importance of CETP in regulating cholesterol homeostasis.     

Muscle atrophy in transgenic mice expressing a human TSC1 transgene

Muscle mass is regulated by a wide range of hormonal and nutritional signals, such as insulin and IGF. Tuberous sclerosis complex (TSC) is an inherited hamartoma disease with tumor growth in numerous organs. TSC is caused by mutation in either TSC1 or TSC2 tumor suppressor genes that negatively regulate insulin-induced S6K activation and cell growth. Here we report that expression of human TSC1 (hTSC1) in mouse skeletal muscle leads to reduction of muscle mass. Expression of hTSC1 stabilizes endogenous TSC2 and leads to inhibition of the mTOR signaling. The hTSC1-mTSC2 hetero-complex and its downstream components remain sensitive to insulin stimulation and nutrition signals. This study suggests that an increase in the steady state level of resident TSC1-TSC2 complex is sufficient to reduce muscle mass and cause atrophy.     

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

We investigated the membrane trafficking of AQP3 induced by epinephrine in Caco-2 cells to clarify the digestive absorption of glycerol permeated by AQP3. Epinephrine was found to promote within 60 min the translocation of AQP3 from the cytoplasmic fraction to the plasma membrane. This increased trafficking of AQP3 was suppressed by phospholipase C and protein kinase C (PKC) inhibitors and a phorbol ester accelerated the trafficking of AQP3 to the membrane fraction. In contrast, adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors did not have any effect on the increased in trafficking of AQP3 by epinephrine and an AC activator did not affect the trafficking of AQP3. Phosphorylation of a threonine (514) residue in PKC was detected upon the treatment with epinephrine and the temporal transitional pattern of this phosphorylation paralleled that of the increased trafficking of AQP3. These results suggest that PKC modulates the trafficking of AQP3.     

Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor

Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.     

Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.     

Generation of a recloned transgenic cat expressing red fluorescence protein

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean = 21 ± 7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications.