Types Of Transgenic Mice Research Articles

Lipin1 prevents ischemic heart disease by regulating lipid homeostasis

Abstract Background Lipid metabolism plays a crucial role in the pathophysiology of cardiovascular diseases. Lipin1 plays a pivotal role in lipid metabolism regulation. In the nucleus, Lipin1 acts as a transcriptional co-stimulatory molecule, interacting with peroxisome proliferator–activated receptor–gamma coactivator 1 alpha (PGC1α) and peroxisome proliferator–activated receptor alpha (PPARα). In the cytoplasm, Lipin1 functions as a phosphatidate phosphatase, converting phosphatidic acid to diacylglycerol to regulate lipid metabolism. Low expression of Lipin1 has been implicated in the severity of cardiac dysfunction in mice. Purpose This study aimed to investigate the role of Lipin1 in the cardiac remodeling after myocardial infarction (MI), revealing its impact on lipid metabolism and molecular pathway associated with cardiac dysfunction. Methods Two types of transgenic mice, cardiomyocyte-specific Lipin1 knockout (cKO) and overexpression (cOE) mice were used in this study. Eight to ten-week-old male mice were subjected to MI by coronary artery ligation. Cardiac lipid droplets were quantified, and triacylglycerol and free fatty acid levels were measured one week after MI surgery. Four weeks after MI, cardiac morphology and function were evaluated using echocardiography, alongside measurements of body weight (BW) and heart weight (HW). Additionally, gene expression analysis and histological evaluation were conducted to assess fatty acid metabolism, cardiac fibrosis, inflammation, and oxidative damage. Results Before MI surgery, there were no differences in HW/BW ratio, left ventricular end-diastolic diameter (LVDd) or LV fractional area change (LVFAC) among cKO/ cOE mice and their littermate controls. After MI, cKO mice exhibited significant cardiac dysfunction with larger LVDd, lower LVFAC and increased HW/BW ratio compared to littermate controls. This was accompanied by exacerbated cardiac fibrosis, increased inflammation, and augmented reactive oxygen species accumulation compared with their controls. Conversely, cOE mice displayed improved LVFAC, reduced cardiac fibrosis, inflammation, and reactive oxygen species accumulation compared to their littermate controls. Notably, the transgene of Lipin1 induced changes in the number of cardiac lipid droplets (LDs). cKO mice hearts showed a reduction in cardiac LDs with decreased levels of triacylglycerol and free fatty acids compared to the littermate controls. In contrast, cOE mice displayed increased cardiac LDs and upregulated expression of fatty acid metabolism–related genes after MI. Conclusion These results suggested that Lipin1 has a protective role against ischemic injury through controlling lipid metabolism in ischemic cardiomyocytes. Thus, Lipin1 emerges as a potential therapeutic target for myocardial infarction.

IMPAQT reveals that adequate mitohormesis from TFAM overexpression leads to life extension in mice.

Mitochondrial transcription factor A, TFAM, is essential for mitochondrial function. We examined the effects of overexpressing the TFAM gene in mice. Two types of transgenic mice were created: TFAM heterozygous (TFAM Tg) and homozygous (TFAM Tg/Tg) mice. TFAM Tg/Tg mice were smaller and leaner notably with longer lifespans. In skeletal muscle, TFAM overexpression changed gene and protein expression in mitochondrial respiratory chain complexes, with down-regulation in complexes 1, 3, and 4 and up-regulation in complexes 2 and 5. The iMPAQT analysis combined with metabolomics was able to clearly separate the metabolomic features of the three types of mice, with increased degradation of fatty acids and branched-chain amino acids and decreased glycolysis in homozygotes. Consistent with these observations, comprehensive gene expression analysis revealed signs of mitochondrial stress, with elevation of genes associated with the integrated and mitochondrial stress responses, including Atf4, Fgf21, and Gdf15. These found that mitohormesis develops and metabolic shifts in skeletal muscle occur as an adaptive strategy.

Unusually Slow Spike Frequency Adaptation in Deep Cerebellar Nuclei Neurons Preserves Linear Transformations on the Subsecond Timescale.

Purkinje cells (PCs) are spontaneously active neurons of the cerebellar cortex that inhibit glutamatergic projection neurons within the deep cerebellar nuclei (DCN) that provide the primary cerebellar output. Brief reductions of PC firing rapidly increase DCN neuron firing. However, prolonged reductions of PC inhibition, as seen in some disease states, certain types of transgenic mice, during optogenetic suppression of PC firing, and in acute slices of the cerebellum, do not lead to large, sustained increases in DCN firing. Here we test whether DCN neurons undergo spike frequency adaptation that could account for these properties. We perform current-clamp recordings at near physiological temperature in acute brain slices from mice of both sexes to examine how DCN neurons respond to prolonged depolarizations. DCN neuron adaptation is exceptionally slow and bidirectional. A depolarizing current step evokes large initial increases in firing that decay to ∼20% of the initial increase within ∼10 s. We find that spike frequency adaptation in DCN neurons is mediated by a novel mechanism that is independent of the most promising candidates, including calcium entry and Na+-activated potassium channels mediated by Slo2.1 and Slo2.2 Slow adaptation allows DCN neurons to gradually and bidirectionally adapt to prolonged currents but to respond linearly to current injection on rapid timescales. This suggests that an important consequence of slow adaptation is that DCN neurons respond linearly to the rate of PC firing on rapid timescales but adapt to slow firing rate changes of PCs on long timescales.SIGNIFICANCE STATEMENT Excitatory neurons in the cerebellar nuclei provide the primary output from the cerebellum. This study finds that these neurons exhibit very slow bidirectional spike frequency adaptation that has important implications for cerebellar function. This mechanism allows neurons in the cerebellar nuclei to adapt to long-lasting changes in synaptic drive while also remaining responsive to short-term changes in excitatory or inhibitory drive.

Gli1+ Cells Residing in Bone Sutures Respond to Mechanical Force via IP3R to Mediate Osteogenesis.

Early orthodontic correction of skeletal malocclusion takes advantage of mechanical force to stimulate unclosed suture remodeling and to promote bone reconstruction; however, the underlying mechanisms remain largely unclear. Gli1+ cells in maxillofacial sutures have been shown to participate in maxillofacial bone development and damage repair. Nevertheless, it remains to be investigated whether these cells participate in mechanical force-induced bone remodeling during orthodontic treatment of skeletal malocclusion. In this study, rapid maxillary expansion (RME) mouse models and mechanical stretch loading cell models were established using two types of transgenic mice which are able to label Gli1+ cells, and we found that Gli1+ cells participated in mechanical force-induced osteogenesis both in vivo and in vitro. Besides, we found mechanical force-induced osteogenesis through inositol 1,4,5-trisphosphate receptor (IP3R), and we observed for the first time that inhibition of Gli1 suppressed an increase in mechanical force-induced IP3R overexpression, suggesting that Gli1+ cells participate in mechanical force-induced osteogenesis through IP3R. Taken together, this study is the first to demonstrate that Gli1+ cells in maxillofacial sutures are involved in mechanical force-induced bone formation through IP3R during orthodontic treatment of skeletal malocclusion. Furthermore, our results provide novel insights regarding the mechanism of orthodontic treatments of skeletal malocclusion.

Abstract 14459: The Cardioprotective Effect on Energy Metabolism of Empagliflozin in Heart of Type 2 Diabetes Model Mouse in vivo

Chronic pathological conditions, such as type 2 diabetes mellitus, involve various mechanisms in promoting heart failure by remodelling energy metabolic pathways and impairing cardiac contractility. The major source of myocardial energetics has been reported to shifts from OXPHOS in normal conditions to glycolysis in heart failure. Therefore, we decided to focus on the effect of empagliflozin on energy metabolic status in the heart.Recently, we generated two types of transgenic mice to monitor energy metabolism, intracellular ATP levels (iATP Tg) and mitochondrial ATP levels (mATP Tg) using FRET biosensor “ATeam” in the whole body, organ, and cellular levels as well as in beating heart. We intercrossed these mice with db/db, a mouse model of type 2 diabetes, and examined the energy metabolism of the heart in the empagliflozin -treated or non-treated groups.db/db;iATP Tg mice were fed EMPA-containing diets (30 mg/kg b.w., day) from 7 weeks of age for 10 weeks, and the ATP levels in the heart were measured by imaging with a fluorescence microscope. The results showed that, unlike the lowered ATP levels in the placebo group, the intracellular ATP level in the heart was significantly increased in the empagliflozin-treated group. Also, the ATP level was recovered in this empagliflozin-treated group to the same level as the wild type.Next, 8 weeks-old db/db;mATP Tg mice received a single dose of empagliflozin (30 mg/kg b.w.) via oral gavage after 4 hr of fasting. After another 3 hr of fasting, monitor the mitochondrial ATP level of the heart in vivo under the fluorescent microscope. The results showed that, unlike the placebo group, the ATP level in the mitochondria of the heart was significantly increased in the empagliflozin-treated group.These results suggest that empagliflozin may restore normal remodelling of energy metabolism in type 2 diabetic hearts.

Animal Models to Study Hepatitis C Virus Infection.

With more than 71 million chronically infected people, the hepatitis C virus (HCV) is a major global health concern. Although new direct acting antivirals have significantly improved the rate of HCV cure, high therapy cost, potential emergence of drug-resistant viral variants, and unavailability of a protective vaccine represent challenges for complete HCV eradication. Relevant animal models are required, and additional development remains necessary, to effectively study HCV biology, virus–host interactions and for the evaluation of new antiviral approaches and prophylactic vaccines. The chimpanzee, the only non-human primate susceptible to experimental HCV infection, has been used extensively to study HCV infection, particularly to analyze the innate and adaptive immune response upon infection. However, financial, practical, and especially ethical constraints have urged the exploration of alternative small animal models. These include different types of transgenic mice, immunodeficient mice of which the liver is engrafted with human hepatocytes (humanized mice) and, more recently, immunocompetent rodents that are susceptible to infection with viruses that are closely related to HCV. In this review, we provide an overview of the currently available animal models that have proven valuable for the study of HCV, and discuss their main benefits and weaknesses.

Animal Tests for Evaluation of Cognitive Impairment in Neonatal Mouse.

For a long time, mice have been less popular than rats for studying cognitive impairment, mainly because much less neuroanatomical and neurochemical information was available on mice than on rats. Over the recent years, the generation of many types of transgenic mice has brought mice to the forefront of this research. Genetically modified mouse models have demonstrated useful to search memory and learning processes and the neurocircuitry and molecular mechanisms involved, as well as to extend therapies for cognitive impairment. A diversity of protocols has been developed to evaluate cognition in mice. The test models have been carefully selected according to reliability of results and disease relevance of the cognitive functions evaluated. Further criteria were ease of application and time efficiency. All tests evaluate slightly different but also interacting aspects or overlapping of learning and memory so that they can be utilized to complement each other in a comprehensive evaluation of cognitive function. In this chapter, three main protocols for evaluation cognitive/behavioral effect induced by drugs in postnatal mouse such as passive avoidance, radial arm maze (RAM), and Morris water maze (MWM) tests are described.

Two-Photon-Excited Fluorescence Microscopy as a Tool to Investigate the Efficacy of Methylprednisolone in a Mouse Spinal Cord Injury Model

Basic imaging experiment. To explore the use of 2-photon-excited fluorescence (2PEF) microscopy to investigate the therapeutic effect of methylprednisolone (MP) in mice with spinal cord injury (SCI). MP can alleviate secondary SCI through its anti-inflammatory effect; however, how MP regulates axonal dynamics in a compression SCI model is not well characterized. We used 2PEF microscopy to trace axonal dynamics in vivo during MP therapy. Two types of transgenic mice (weighing 23-25 g) including YFP-H line (n = 18) and CX3CR1-GFP (n = 18) were used for experimental procedure. Each type of mouse was randomly divided into 3 groups, and the sample size of every subgroup was 6. The sham groups including YFP-H line group (n = 6) and CX3CR1-GFP group (n = 6) received laminectomy only (group 1). SCI groups received saline treatment (group 2) and SCI groups received MP treatment (group 3). Hind limb motor function was evaluated using the Basso Mouse Scale. 2PEF microscopy was used to image in vivo axonal dynamics at baseline and at 0.5 hours, 24 hours, 48 hours, and 72 hours postinjury. Histology was employed to examine pathological changes and microglial/macrophage proliferation after all imaging sessions. Group 1 exhibited no significant differences in hind limb motor function before versus after surgery. The Basso Mouse Scale scores were significantly lower in groups 2 and 3 than in group 1 (P < 0.05). Degree of recovery was higher in group 3 than in group 2 at 7 days postinjury (P < 0.05). The axons in group 1 remained intact at all time points. The survival rate of axons in groups 2 and 3 progressively decreased at 48 hours postinjury; at 72 hours postinjury, the axon survival rate was higher in group 3 than group 2 (P < 0.05). Histology revealed that group 3 presented milder damage in injured spinal cord than group 2. Microglial/macrophage proliferation was lower in group 3 than in group 2 (P < 0.05). 2PEF microscopy is useful for detecting early changes, indicating axonal disruption in compression SCI. MP therapy may help alleviate axonal progressive damage and reduce the proliferation of microglia/macrophages in acute SCI. N/A.

Enhanced Cancer Radiotherapy through Immunosuppressive Stromal Cell Destruction in Tumors

Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs. Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing. We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines. These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers.

Sleep–wake characterization of double MT1/MT2 receptor knockout mice and comparison with MT1 and MT2 receptor knockout mice

The neurohormone melatonin activates two G-protein coupled receptors, MT1 and MT2. Melatonin is implicated in circadian rhythms and sleep regulation, but the role of its receptors remains to be defined. We have therefore characterized the spontaneous vigilance states in wild-type (WT) mice and in three different types of transgenic mice: mice with genetic inactivation of MT1 (MT1−/−), MT2 (MT2−/−) and both MT1/MT2 (MT1−/−/MT2−/−) receptors.Electroencephalographic (EEG) and electromyographic sleep–wake patterns were recorded across the 24-h light–dark cycle. MT1−/−mice displayed a decrease (−37.3%) of the 24-h rapid eye movement sleep (REMS) time whereas MT2−/−mice showed a decrease (−17.3%) of the 24-h non rapid eye movement sleep (NREMS) time and an increase in wakefulness time (14.8%). These differences were the result of changes occurring in particular during the light/inactive phase. Surprisingly, MT1−/−/MT2−/− mice showed only an increase (8.9%) of the time spent awake during the 24-h. These changes were correlated to a decrease of the REMS EEG theta power in MT1−/−mice, of the NREMS EEG delta power in MT2−/−mice, and an increase of the REMS and wakefulness EEG theta power in MT1−/−/MT2−/− mice. Our results show that the genetic inactivation of both MT1 and MT2 receptors produces an increase of wakefulness, likely as a result of reduced NREMS due to the lack of MT2 receptors, and reduced REMS induced by the lack of MT1 receptors. Therefore, each melatonin receptor subtype differently regulates the vigilance states: MT2 receptors mainly NREMS, whereas MT1 receptors REMS.

Transgenic mice: beyond the knockout

Transgenic mice have had a tremendous impact on biomedical research. Most researchers are familiar with transgenic mice that carry Cre recombinase (Cre) and how they are used to create conditional knockouts. However, some researchers are less familiar with many of the other types of transgenic mice and their applications. For example, transgenic mice can be used to study biochemical and molecular pathways in primary cultures and cell suspensions derived from transgenic mice, cell-cell interactions using multiple fluorescent proteins in the same mouse, and the cell cycle in real time and in the whole animal, and they can be used to perform deep tissue imaging in the whole animal, follow cell lineage during development and disease, and isolate large quantities of a pure cell type directly from organs. These novel transgenic mice and their applications provide the means for studying of molecular and biochemical events in the whole animal that was previously limited to cell cultures. In conclusion, transgenic mice are not just for generating knockouts.

The use of transgenic fluorescent mouse strains, fluorescent protein coding vectors, and innovative imaging techniques in the life sciences

paper by Fujiki et al. entitled ‘‘Quantification of greenfluorescence protein by in vivo imaging, PCR and flow cyto-metry: comparison of transgenic strains and relevance for fetalcell microchimerism’’ has a very promising approach to imagefetal cell microchimerism (1). The paper compares two typesof transgenic mice expressing green fluorescent protein (GFP)for the future study of fetal microchimerism—the processwhere fetal cells enter the maternal circulation, and in humanscan persist in the maternal blood and tissues for decades. TheGFP transgenic mice chosen for study are the C51B6/6-Tg(ACTB-EGFP) Osb/J (CAG) and the ROSA 26-EGFP. Theauthors conclude that the CAG mice have much brighter cells,but the ROSA 26-EGFP cells more consistently express GFP.The authors compare imaging, PCR, and flow cytometry(FCM) to image cells from these mice and conclude that flowcytometry is the method of choice, since ‘‘. . . due to the exist-ing status of this imaging technology and the purely quantita-tive nature of PCR amplification, FCM provides the best over-all approach for the quantitative and qualitative evaluation ofrare transgenic microchimeric fetal cells.’’The authors, however, are apparently unaware of thepower of fluorescence protein-imaging in vivo. Chishima et al.(2) pioneered this area and showed that single cancer cellsexpressing GFP could be readily imaged in autopsied miceagainst the large background of normal tissue. Subsequently,Yang et al. (3) showed that tumors could be whole-bodyimaged with GFP in live unrestrained mice. Yang et al. (4)then made two more important advances, and showed in skinflaps in live mice that single GFP cells could be imaged inmany organs. Yang et al. (5) then showed that tumor cellsexpressing red fluorescent protein (RFP) in the cytoplasm andGFP in the nucleus, as well as GFP host stromal cells, couldnoninvasively be imaged when grown in the mouse foot pad.Yamamoto et al. (6) and Yamauchi et al. (7,8) showedthat single cells labeled in the nucleus with GFP and in thecytoplasm with RFP could be imaged in mice both noninva-sively as well as in skin flaps. Yamauchi et al. (7,8) imaged thedual color cells in blood vessels as they trafficked in real time.Imaging cell trafficking was greatly enhanced by use of thevariable-magnification Olympus OV100 imaging system.Hayashi et al. (9) used the dual-colored cells and OlympusOV100 to image cells trafficking in lymphatics.Use of a high-powered variable-magnification imagingsystem, such as the OV100, rather than the IVIS 200 photoncounting device used by Fujiki et al. which does not produceimages, enables single fluorescent-protein-labeled cells to bevisualized in real time even during trafficking (10–12). Suchan approach offers many advantages over the IVIS 200 andflow cytometry, since cells can be visualized in the live animalwhich is very important for studying microchimerism (13).In parallel to the rapid development of instrumentation,many efforts have been made to optimize the existing ‘‘classical’’

On the use of the T‐REx™ tetracycline‐inducible gene expression system in vivo

Components of the commercially available T-REx system were used to create two types of transgenic mice. The first contained the tetracycline-repressor transgene under the control of the CMV promoter/enhancer; the second type contained a green fluorescent protein (GFP) reporter transgene under the control of the CMV promoter/enhancer with a tetracycline repressor operator sequence. Transgene expression was unpredictable in animals containing the individual transgenes. Animals with the reporter transgene expressed GFP in only some tissues (e.g., pancreas, kidney), and one line of reporter transgenic animals developed kidney disease, presumably due to expression of the transgene. The two types of transgenic animals were crossbred to produce double-transgenic animals with the object of regulating the expression of the reporter in vivo. When a similar double-transgenic system was constructed in cultured cells, the repressor protein suppressed the transcription of the reporter transgene. The presence of the repressor in double-transgenic animals had no effect on the expression of the reporter; double transgenic animals developed the same kidney disease that was seen in singly transgenic mice with the reporter. Our results indicate that transgenes under the control of the CMV promoter in the T-REx system express somewhat unpredictably and in only a limited number of tissues, making the use of this system for the development of in vivo models problematical.

Stratum-Specific Expression of Human Transferrin Receptor Increases Iron in Mouse Epidermis

Epidermal desquamation accounts for 20% of the body's iron loss each day. Yet, little is known about how iron content in epidermis is regulated. To test the importance of the transferrin receptor in regulating iron content in epidermis, we created transgenic mice that have stratum-specific expression of the human transferrin receptor. The keratin 14 promoter targeted the receptor primarily to basal, proliferating keratinocytes; the involucrin (Inv) promoter targeted the receptor to suprabasal, differentiating keratinocytes. There were age- and site-dependent differences in iron content in the epidermis and hair. In both types of transgenic mice, epidermal iron content increased with age and at 8 weeks was 2-3-fold greater in transgenic mice compared to littermate controls. Iron was increased up to 2-fold in hair of keratin 14-human transferrin receptor (hTfR) transgenics and 30% in Inv-hTfR transgenics. No gross or histological changes were seen in transgenic animals with increased iron in the epidermis. Ferritin expression, which was low in normal epidermis, was greatly increased in both transgenic lines, indicating that it is the likely depot for the extra iron in these animals. These data show that control of transferrin receptor expression is sufficient to regulate iron content in proliferating or differentiating keratinocytes in the epidermis.

NKG2D in Tumor and Graft Surveillance

Natural killer (NK) cells are involved in graft rejection and may contribute to tumor recognition and elimination. Ogasawara et al. report that increased production of NKG2D ligands may be one mechanism by which certain tissues are prone to graft rejection and that antibodies against NKGD2 can prevent rejection in these cases. Oppenheim et al. report that localized high expression of NKG2D ligands inhibited NK cell function, contributing to evasion of the immune system by tumors. To study graft rejection, Ogasawara et al. evaluated NKG2D ligand expression (by antibody staining) of bone marrow cells from BALB/C or C57BL/6 mice that were transplanted into irradiated F 1 progeny from a BALB/C C57BL/6 cross, an example of F 1 hybrid resistance. The BALB/C cells expressed the ligand Rae-1 and were rejected by the F1 progeny mice. The C57BL/6 mouse cells were also rejected, but they did not express any NKG2D ligands; thus, there are at least two mechanisms for NK cell-mediated rejection. Depletion of the NK cells from the recipient mice allowed the transplanted cells from both parental strains to survive; however, only in the case of the BALB/C transplanted cells did neutralizing antibodies to NKG2D increase the incorporation of the transplanted cells in the spleens of the recipient mice. The Rae-1 ligand was also the focus of the studies by Oppenheim et al. In this case, two kinds of transgenic mice were generated: those expressing Rae-1ε in squamous epithelium only (lines 110 and 121) and those expressing Rae-1ε ubiquitously (line 187). Both types of transgenic mice showed decreased abundance of NKG2D-positive NK cells; however, the abundance of other proteins (such as CD94) typically expressed by this population of NK cells was normal. Thus, the NKG2D receptor itself appeared to be down-regulated rather than the cell population depleted. Coculturing of NKG2D-positive splenocytes with the splenocytes from the 187 line (high Rae-11ε) resulted in loss of NKG2D from the nontransgenic cells. Injection of a mixture of splenocytes from the 187 line and control splenocytes into control mice or transgenic mice showed that clearance of the Rae-1-positive cells was impaired in the transgenic mice. Activation of the NK cells with poly (I:C) restored killing of the Rae-1-positive cells in the epithelial transgenic mice only, suggesting that a residual population of NKG2D-positive cells could be rescued in the 110 and 121 lines. Finally, the transgenic mice showed increased susceptibility to tumorigenesis (chemical induced and injection of tumor cells). Thus, the NKG2D receptor is a critical player in tumor surveillance and graft rejection. K. Ogasawara, J. Benjamin, R. Takaki, J. H. Phillips, L. L. Lanier, Function of NKG2D in natural killer cell-mediated rejection of mouse bone marrow grafts. Nat. Immunol. 6 , 938-945 (2005). [Online Journal] D. E. Oppenheim, S. J. Roberts, S. L. Clarke, R. Filler, J. M. Lewis, R. E. Tigelaar, M. Girardi, A. C. Hayday, Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor surveillance. Nat. Immunol. 6 , 928-937 (2005). [Online Journal]

Analysis of Rat Insulin II Promoter-Ghrelin Transgenic Mice and Rat Glucagon Promoter-Ghrelin Transgenic Mice

We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, beta cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.

Transgenic expression of Korean type hepatitis C virus core protein and related mutants in mice

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S1161, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S1161 transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism.

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.

Overexpression of heat shock protein 70 in R6/2 Huntington’s disease mice has only modest effects on disease progression

Huntington’s disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine tract in a protein called huntingtin. The inducible form of heat shock protein 70 (Hsp70) has been shown to reduce polyglutamine-induced toxicity. To investigate if overexpression of Hsp70 can affect disease progression in a mouse model of HD, we crossed R6/2 mice, expressing exon 1 of the HD gene with an expanded CAG repeat, with mice overexpressing Hsp70 (both types of transgenic mice were of the CBAxC57BL/6 strain). The resulting R6/2-Hsp70 transgenics exhibited 5- to 15-fold increases in Hsp70 expression in neocortical, hippocampal and basal ganglia regions. This correlated with a delayed loss of body weight compared to R6/2 mice. However, the number or size of nuclear inclusions, the loss of brain weight, reduction of striatal volume, reduction in size of striatal projection neurons, downregulation of DARPP-32, development of paw clasping phenotype and early death of the mice were not affected by Hsp70 overexpression. Interestingly, the polyglutamine protein affected the potential rescuing agent, because in older R6/2-Hsp70 mice a large proportion of the Hsp70 protein was sequestrated in nuclear inclusions.

Both Donor and Recipient Origins of Smooth Muscle Cells in Vein Graft Atherosclerotic Lesions

Smooth muscle cell (SMC) accumulation in the inner layer of the vessel wall is a key event in the pathogenesis of atherosclerosis in vein grafts, but the origin of the cells in these lesions has yet to be shown. Herein, we use animal models of vein grafts in transgenic mice to clearly identify the sources of SMCs in atherosclerosis. Vena cava segments were isografted to carotid arteries between four types of transgenic mice, including SM-LacZ expressing beta-galactosidase (beta-gal) in vascular SMCs, SM-LacZ/apoE(-/-), ROSA26 expressing beta-gal in all tissues, and wild-type mice. beta-gal-positive cells were observed in neointimal and atherosclerotic lesions of all vein segments grafted between LacZ transgenic and wild-type mice. Double staining for beta-gal and cell nuclei revealed that about 40% of SMCs originated from hosts and 60% from the donor vessel. This was confirmed by double labeling of the Y-chromosome and alpha-actin in the lesions of sex-mismatched vein grafts. The possibility that bone marrow cells were the source of SMCs in grafts was eliminated by the absence of beta-gal staining in atherosclerotic lesions of chimeric mice. Furthermore, vein SMCs of SM-LacZ mice did not express beta-gal in situ, but did so when these cells appeared in atherosclerotic lesions in vivo, suggesting that hemodynamic forces may be crucial for SMC differentiation. Thus, we provide the first evidence of SMC origins in the atherosclerotic lesions of vein grafts, which will be essential for providing insight into new types of therapy for the disease. The full text of this article is available at http://www.circresaha.org.