Tau filament formation in transgenic mice expressing P301L tau

Mutations in the microtubule-associated protein tau, including P301L, are genetically coupled to hereditary frontotemporal dementia with parkinsonism linked to chromosome 17. To determine whether P301L is associated with fibril formation in mice, we expressed the longest human tau isoform, human tau40, with this mutation in transgenic mice by using the neuron-specific mouse Thy1.2 promoter. We obtained mice with high expression of human P301L tau in cortical and hippocampal neurons. Accumulated tau was hyperphosphorylated and translocated from axonal to somatodendritic compartments and was accompanied by astrocytosis and neuronal apoptosis indicated by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling staining. Moreover, P301L tau formed abnormal filaments. Electron microscopy of sarcosyl-insoluble protein extracts established that the filaments had a straight or twisted structure of variable length and were approximately 15 nm wide. Immunoelcecton microscopy showed that the tau filaments were phosphorylated at the TG3, AT100, AT8, and AD199 epitopes in vivo. In cortex, brain stem, and spinal cord, neurofibrillary tangles were also identified by thioflavin-S fluorescent microscopy and Gallyas silver stains. Together, our results show that expression of the P301L mutation in mice causes neuronal lesions that are similar to those seen in human tauopathies.     

Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.     

Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility

A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.     

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.     

Relationship between simian virus 40 large tumor antigen expression and tumor formation in transgenic mice.

A line of transgenic mice containing the simian virus 40 (SV40) large tumor antigen gene under the control of the viral enhancer-promoter expressed this viral protein in the brains of these mice within the first 2 weeks after birth. Multiple foci of anaplastic cells formed in the choroid plexuses of these mice at 36 to 41 days after birth, and normal tissue coexisted with these transformed foci. Immunoperoxidase staining to detect the SV40 T antigen showed tumor-specific expression of nuclear T antigen at late times in tumor development, approximately 90 to 100 days and thereafter. The level of SV40 T antigen, on a per cell basis, appeared to be lower in the great majority of choroid plexus cells at earlier times in tumor development. These results suggest that low levels of tumor antigen (14 to 36 days) are present before detectable pathology (36 to 41 days) and the level of T antigen per cell is higher in rapidly growing late-stage tumors (older than 90 days).     

绿色荧光蛋白转基因小鼠模型的建立及胚胎冷冻保种

目的建立绿色荧光蛋白转基因小鼠模型,并采取胚胎冷冻的方法进行保种。方法通过原核显微注射法,把线性化、纯化后的外源基因pEGFP注射入BDF1小鼠受精卵中,胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。经鉴定对有表达的转基因鼠进行胚胎冷冻保种。结果移植注射胚胎385枚给30只假孕小鼠共出生了306只后代鼠,经PCR和southern blot检测得到5只阳性小鼠。F2代转基因鼠胚胎冷冻240枚胚胎。结论通过显微注射法使外源基因pEGFP在小鼠基因组中得到整合,建立了转pEGFP的转基因小鼠模型。     

Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter.

The promoter region of the suprabasal keratin 10 gene has been used to direct expression of a mutant human Harvey-ras oncogene to the differentiating cells of the mouse epidermis. Transgenic animals develop hyperkeratosis of the skin and forestomach--the two sites known to express high levels of the keratin 10 polypeptide in vivo. Papillomas subsequently develop on the skin surface, initially at sites subject to biting or scratching such as the base of the tail or behind the ears. The results suggest that the "second event" involved in tumor development in these transgenic animals is the local induction of a mild wounding stimulus. Furthermore, because the H-ras transgene is expressed in suprabasal cells, it appears that cells which have left the stem cell compartment can be induced to form at least benign tumors in vivo.     

Enhanced shutoff of phototransduction in transgenic mice expressing palmitoylation-deficient rhodopsin

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.     

Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.

Spontaneous intestinal and intra-abdominal bleeding was observed in a high percentage of newborn transgenic mice carrying the murine urokinase-type plasminogen activator (uPA) gene linked to the albumin enhancer/promoter. These hemorrhagic events were directly related to transgene expression in the liver and the development of high plasma uPA levels. Two lines were established from surviving founder mice that displayed multigenerational transmission of the bleeding phenotype. Fatal hemorrhaging developed between 3 and 84 hr after birth in about half of the transgenic offspring of these lines; transgenic pups that did not bleed nevertheless passed the phenotype to their young. The phenotypic variability could not be explained by differences in transgene expression. All transgenic neonates were severely hypofibrinogenemic and displayed loss of clotting function that extended beyond the risk period for bleeding. These mice provide a means of studying the pathophysiology of plasminogen hyperactivation and evaluating therapeutic protocols designed to prevent bleeding.     

Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C

The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.     

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

Background  

Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice.     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.     

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.     

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.     

Mammary tumor heterogeneity in wt-ErbB-2 transgenic mice

Phenotypic and biological heterogeneity was studied in a single transgenic mouse model to determine the level of biological variance. We analyzed 1,258 tumors from 417 MMTV-wt-ErbB-2 transgenic mice, subdivided by casein or soy-based dietary randomization and hormonal treatment. Variance in tumor histologic features, growth pattern, invasion, metastases, and multi-focality were detected in untreated and treated mice. Ninety-three percent (1,174/1,258) of tumors had the solid growth pattern widely reported in this model. However, among the solid tumors, a spectrum of growth patterns, from well-circumscribed tumors with a pseudocapsule to locally invasive or highly aggressive, metastatic subtype, was observed. Of the non-solid tumors, glandular features were prominent in 84 (7%). Adenocarcinomas included papillary, acinar/glandular, and adenosquamous subtypes. Adenosquamous tumors were exclusively observed in the group of mice treated on a short-term basis with estrogen. In contrast to the reported literature for this transgenic mouse model, mammary tumors were multifocal in the majority of cases (303 of 417 mice, or 73%). Results of this extensive study of a single transgenic model of mammary tumorigenesis indicate phenotypic and biological heterogeneity not previously associated with this transgenic mouse. These data support a complex, multistep process of carcinogenesis and clonal evolution, with biological and phenotypic variance similar to that observed in human mammary cancer development.