Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.     

Ectopic expression of Gcm1 induces congenital spinal cord abnormalities

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.     

Ectopic expression of Hox-2.3 induces craniofacial and skeletal malformations in transgenic mice.

To better understand the role of the Hox-2.3 murine homeobox gene during development, a dominant gain-of-function mutation was generated. The developmental malformations that resulted when the chicken beta-actin promoter was used to direct widespread expression of the Hox-2.3 gene in transgenic mice included early postnatal death as well as craniofacial abnormalities, including open eyes and cleft palate. Ventricular septal defects were also observed in the hearts of three transgenic mice. Skeletal malformations were seen in the bones of the craniocervical transition, with the occipital, basisphenoid, and atlas bones deficient or misshapen. Interestingly, one mutant exhibited an extra pair of ribs as well as alterations in cervical vertebrae identities. Some of the malformations observed in Hox-2.3 gain-of-function mutants overlap with those seen in Hox-1.1 and Hox-2.2 misexpression mutants which suggests functional similarities between paralogous homeobox genes. The results of these experiments are consistent with a role for Hox-2.3 in specifying positional information during development.     

T cell independent Thy-1 allo-antibody response with the use of transgenic mice

We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.     

Morphometric and functional abnormalities of kidneys in the progeny of mice fed chocolate during pregnancy and lactation

Even most commonly consumed beverages like tea, coffee, chocolate and cocoa contain methylxanthines, biogenic amines and polyphenols, among them catechins, that exhibit significant biological activity and might profoundly affect the organism homeostasis. We have previously shown that 400 mg of bitter chocolate or 6 mg of theobromine added to the daily diet of pregnant and afterwards lactating mice affected embryonic angiogenesis and caused bone mineralization disturbances as well as limb shortening in 4-weeks old offspring. The aim of the present study was the morphometric and functional evaluation of kidneys in the 4-weeks old progeny mice fed according to the protocol mentioned above. Progeny from the mice fed chocolate presented considerable morphometric abnormalities in the kidney structure, with the lower number of glomeruli per mm2 and their increased diameter. Moreover, higher serum creatinine concentration was observed in that group of offspring. No morphometric or functional irregularities were found in the progeny of mice fed theobromine. Abnormalities demonstrated in the offspring of mice fed chocolate are not related to its theobromine content. Consequently, identification of active compound(s) responsible for the observed effects is of vital importance.     

Ectopic expression of soybean leghemoglobin in chloroplasts impairs gibberellin biosynthesis and induces dwarfism in transgenic potato plants

We have characterized potato (Solanum tuberosum L.) plants expressing a soybean leghemoglobin that is targeted to plastids. Transgenic plants displayed a dwarf phenotype caused by short internode length, and exhibited increased tuberization in vitro. Under in vivo conditions that do not promote tuberization, plants showed smaller parenchymal cells than control plants. Analysis of gibberellin (GA) concentrations indicated that the transgenic plants have a substantial reduction (approximately 10-fold) of bioactive GA₁ concentration in shoots. Application of GA₃ to the shoot apex of the transformed plants completely restored the wild type phenotype suggesting that GA-biosynthesis rather than signal transduction was limiting. Since the first stage of the GA-biosynthetic pathway is located in the plastid, these results suggest that an early step in the pathway may be affected by the presence of the leghemoglobin.     

Cross-contamination with tamoxifen induces transgene expression in non-exposed inducible transgenic mice

Inducible transgenic mouse models that impose a constraint on both temporal and spatial expression of a given transgene are invaluable. These animals facilitate experiments that can address the role of a specific cell or group of cells within an animal or in a particular window of time. A common approach to achieve inducibility involves the site-specific recombinase 'Cre', which is linked to a modified version of one of various steroid hormone-binding domains. Thus, the expression of Cre is regulated such that a functional nuclear transgene product can only be generated with the addition of an exogenous ligand. However, critical requirements of this system are that the nuclear localization of the transgene product be tightly regulated, that the dosage of the inducing agent remains consistent among experimental animals and that the transgene cassette cannot express in the absence of the inducing agent. We used the Cre ER(T2) cassette, which is regulated by the addition of the estrogen antagonist tamoxifen to determine whether cross-contamination of tamoxifen between animals housed together can be a significant source of spurious results. We found that cross-contamination of exogenous tamoxifen does occur. It occurred in all animals tested. We suggest that the mechanism of contamination is through exposure to tamoxifen in the general environment and/or to coprophagous behavior. These results have important implications for the interpretation and design of experiments that use 'inducible' transgenic animals.     

Ubiquitous expression of mRFP1 in transgenic mice

Fluorescent proteins provide a powerful means to track gene expression and cellular behaviors in the study of model organisms such as mice. Among the new generation of fluorescent protein markers, the monomeric red fluorescent protein mRFP1 is particularly attractive because of its rapid maturation and minimal interference with GFP and GFP-derived markers. Here we evaluate the utility of mRFP1 as a marker in transgenic mice. We show that high level and ubiquitous expression of mRFP1 does not affect mouse development, general physiology, or reproduction. mRFP1 expression can be readily detected with unaided eyes under daylight in transgenic mice on the albino background. The intensity of mRFP1 signals can be used to distinguish homozygous and heterozygous transgenic mice. Together, these features make mRFP1 an attractive marker for broad applications in transgenic research.     

Ectopic expression of desmin in the epidermis of transgenic mice permits development of a normal epidermis

Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.     

Ectopic expression of gamma interferon in the eye protects transgenic mice from intraocular herpes simplex virus type 1 infections.

Transgenic (rho gamma) mice provide a model for studying the influence of gamma interferon (IFN-gamma) produced in the eye on ocular and cerebral viral infection. To establish this model, we injected BALB/c- and C57BL/6-derived transgenic and nontransgenic mice of different ages intravitreally with herpes simplex virus type 1 (HSV-1) strain F. Eye and brain tissues of these mice were assessed for pathological and immunocytochemical changes. HSV-1 infection induced severe retinitis of the injected eyes and infection of the brain in all mice. In transgenic mice inoculated with HSV-1, the left, nontreated eyes were protected from retinitis, whereas nontransgenic mice developed bilateral retinitis. Additional intravitreal injection of IFN-gamma with the virus protected the noninoculated eyes of nontransgenic mice. Three-week-old nontransgenic mice died from HSV-1 infection, whereas transgenic mice of the same age and nontransgenic mice intravitreally treated with IFN-gamma survived. Ocular IFN-gamma production increased the extent of inflammation in transgenic mice but did not have a significant influence on the growth of HSV-1 until day 3 after inoculation and did not influence the neuroinvasion of this virus. Thus, the effects of IFN-gamma were not caused by an early block of viral replication. Possible mechanisms of IFN-gamma action include activation of the immune response, alteration of the properties of the virus, and direct protection of neurons.     

Vasopressin-SV40 T antigen expression in transgenic mice induces brain tumor and lymphoma

In order to understand the importance of various cis-acting elements in regulating VP gene expression, transgenic mice regulated by VP constructs were produced containing 3.8 kb of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to an SV40 T antigen (Tag). In the transgenic mice by the pVPSV.IGR3.6 construct, all the six transgenic mice died at the age of 2-6 weeks. In the transgenic mice by pVPSV.IGR2.1, 21% of them had brain tumors at 5 weeks and 100% of the mice had brain tumors after 24 weeks. Histological analysis of the transgenic mice revealed primitive neuroectodermal tumors (PNET) in the brain and lymphoma in the spleen and lymph nodes. The phenotype differences between the two transgenic mice suggest that tissue-specific expression might be regulated by cis-acting elements in the 1.5-kb of the 3(') flanking region, which are not contained in pVPSV.IGR2.1. In conclusion, pVPSV.IGR2.1 mice will be a valuable mouse model system for investigating PNET tumorigenesis in the brain and lymphoma in the lymph nodes and spleen.     

Expression of an epidermal keratin protein in liver of transgenic mice causes structural and functional abnormalities

To examine the role of keratin intermediate filament proteins in cell structure and function, transgenic mice were isolated that express a modified form of the human K14 keratin protein in liver hepatocytes. A modified K14 cDNA (K14.P) sequence was linked downstream of the mouse transthyretin (TTR) gene promoter and enhancer elements to achieve targeted expression in hepatocytes. Hepatocytes expressing high levels of the transgene were found to have abnormal keratin filament networks as detected by indirect immunofluorescence using an antibody specific for the transgene product. Light and electron microscopic level histological analysis of isolated liver tissue showed in many cases degenerative changes that included inflammatory infiltration, ballooning degeneration, an increase in fat containing vacuoles, and glycogen accumulation. These changes were most evident in older mice over four months of age. No indication of typical Mallory body structures were identified at either the light or electron microscopic level. To evaluate secretory function in transgenic livers, bile acid secretion rates were measured in isolated perfused liver and found to be approximately twofold lower than aged-matched controls. These findings indicate that expression of an abnormal keratin in liver epithelial cells in the in vivo setting can alter the structure and function of a tissue and suggest a role of the keratin network in cellular secretion.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

Ectopic expression of KNOTTED1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice

Some phytohormones such as gibberellins (GAs) and cytokinins (CKs) are potential targets of the KNOTTED1-like homeobox (KNOX) protein. To enhance our understanding of KNOX protein function in plant development, we identified rice (Oryza sativa) genes for adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CK biosynthesis. Molecular and biochemical studies revealed that there are eight IPT genes, OsIPT1 to OsIPT8, in the rice genome, including a pseudogene, OsIPT6. Overexpression of OsIPTs in transgenic rice inhibited root development and promoted axillary bud growth, indicating that OsIPTs are functional in vivo. Phenotypes of OsIPT overexpressers resembled those of KNOX-overproducing transgenic rice, although OsIPT overexpressers did not form roots or ectopic meristems, both of which are observed in KNOX overproducers. Expression of two OsIPT genes, OsIPT2 and OsIPT3, was up-regulated in response to the induction of KNOX protein function with similar kinetics to those of down-regulation of GA 20-oxidase genes, target genes of KNOX proteins in dicots. However, expression of these two OsIPT genes was not regulated in a feedback manner. These results suggest that OsIPT2 and OsIPT3 have unique roles in the developmental process, which is controlled by KNOX proteins, rather than in the maintenance of bioactive CK levels in rice. On the basis of these findings, we concluded that KNOX protein simultaneously decreases GA biosynthesis and increases de novo CK biosynthesis through the induction of OsIPT2 and OsIPT3 expression, and the resulting high-CK and low-GA condition is required for formation and maintenance of the meristem.