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The promoter of the GABA(A) receptor delta subunit gene was analyzed in transgenic mice and in cultured cells to study sequences involved in neuronal subtype-specific gene expression. A 6.4-kb genomic fragment faithfully directed neuron-specific transcription of a lacZ reporter gene in the central nervous system. The transgene expression pattern in the cerebral cortex, hippocampal formation, thalamus, and brainstem was consistent with the regional and neuronal subtype-specific expression of the endogenous delta subunit protein in both developing and mature brain. In the cerebellum, however, the transgene was ectopically expressed in Purkinje cells and silent in granule cells, where the endogenous delta subunit is abundantly expressed. These mice provide a useful tool for investigating activity-dependent regulation of GABA(A) receptor expression under physiological and pathological conditions. Transfection studies using primary cortical neurons and astroglial cells revealed that the delta subunit gene promoter was selectively active in neurons even when truncated to a 267-bp core fragment. In conclusion, the delta subunit gene promoter/upstream region contains the information for neuronal subtype-specific expression in the entire brain except in the cerebellum and is selectively active in primary cortical neurons in vitro.

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.

We have generated a transgenic mouse model for astrocytoma by expressing the v-src kinase under control of the glial fibrillary acidic protein (GFAP) gene regulatory elements in astrocytes. Abnormal astrogliosis was observed in all transgenic animals already at 2 weeks postnatally, frequently followed by the development of dysplastic changes. Later, small proliferative foci arose, and overt astrocytoma developed in the brain and spinal cord in 14.4% of mice after a follow up time of 65 weeks. While early lesions were histologically consistent with low-grade astrocytoma, at later stages most tumors were highly mitotic and frankly malignant. Vascular endothelial growth factor (VEGF) was expressed by tumor cells already at early stages, suggesting induction by v-src, and it was most pronounced in pseudopalisading cells surrounding necrotic areas, implying additional upregulation by hypoxia. In larger lesions, mitotic activity and expression of flk-1, the cognate receptor of VEGF were induced in endothelial cells. Therefore, end-stage tumors mimicked the morphological and molecular characteristics of human glioblastoma multiforme. Time course and stochastic nature of the process indicate that v-src did not suffice for malignant transformation, and that astrocytomas were the result of a multistep process necessitating co-operation of additional genetic events.

One evolutionary explanation for the success of sexual reproduction assumes that sex is an advantage in the coevolutionary arms race between pathogens and hosts. Accordingly, an important criterion in mate choice and maternal selection thereafter could be the allelic specificity at polymorphic loci involved in parasite-host interactions, e.g. the MHC (major histocompatibility complex). The MHC has been found to influence mate choice and selective abortions in mice and humans. However, it could also influence the fertilization process itself, i.e. (i) the oocyte's choice for the fertilizing sperm, and (ii) the outcome of the second meiotic division after the sperm has entered the egg. We tested both hypotheses in an in vitro fertilization experiment with two inbred mouse strains congenic for their MHC. The genotypes of the resulting blastocysts were determined by polymerase chain reaction. We found nonrandom MHC combinations in the blastocysts which may result from both possible choice mechanisms. The outcome changed significantly over time, indicating that a choice for MHC combinations during fertilization may be influenced by one or several external factors.

The amyloid precursor protein (beta APP) gene of the mouse was disrupted by homologous recombination; however, contrary to expectation, brain and other tissues still contained beta APP-specific RNA, albeit at a level 5-10 fold lower than wild-type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4', 6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

During most immune responses, T cells help antigen-specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the beta chain (V beta 2) of a major histocompatibility complex class II-restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV-IND). These mice exhibit an increased precursor frequency of VSV-specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching.

Fragile X syndrome is one of the most common genetic causes of mental retardation, yet the mechanisms controlling expression of the fragile X mental retardation gene FMR1 are poorly understood. To identify sequences regulating FMR1 transcription, transgenic mouse lines were established using a fusion gene consisting of an E.coli beta-galactosidase reporter gene (lacZ) linked to a 2.8 kb fragment spanning the 5'-region of FMR1. Five transgenic mouse lines showed lacZ expression in brain, in particular in neurons of the hippocampus and the granular layer of the cerebellum. Expression of the reporter gene was also detected in Leydig cells and spermatogonia in the testis, in many epithelia of adult mice, and in the two other steroidogenic cell types, adrenal cortex cells and ovarian follicle cells. Embryonic tissues which showed strong activity of the reporter gene included the telencephalon, the genital ridge, and the notochord. This expression pattern closely resembles the endogenous one, indicating that the 5' FMR1 gene promoter region used in this study contains most cis-acting elements regulating FMR1 transcription.

Transgenic mice expressing rat parvalbumin under the control of the human metallothionein IIA (MTII A), SV-40 early, and neuron-specific enolase (NSE) promoters were produced. Ectopic expression was analyzed by RNA polymerase chain reaction and RNase protection in combination with immunohistochemistry. From a total of 25 transgenic lines 18 were found to express the transgene. Expression strength and tissue specificity were dependent upon the promoter used and varied considerably among animal lines produced with the same construct. Highest constitutive MT IIA-driven expression was found in lung, liver, heart, and kidney, as well as in brain, and lower amounts of transgene expression were found in spleen, testis, and muscle. Immunohistochemistry of tissue sections of metallothionein-parvalbumin transgenic strain 29 in the non-induced state revealed that ectopic PV mRNA is translated into protein. Short-term induction of the MT IIA promoter by CdSO4 or CdCl2 leads to a shift in tissue specificity and does not increase ectopic expression in tissues where the transgene is active in the noninduced state. As expected the NSE promoter showed highest activity in brain. However, NSE-driven expression could also be detected to various degrees in all investigated tissues. SV-40-dependent PV expression showed no tissue preference and varied considerably among different strains. Except for the observation that the SV-40-PV construct showed lower yields in transgenic production and reduced numbers of positive offspring no obvious impairment of growth or behavior as a consequence of transgenic PV expression could be detected.

The beta-amyloid precursor protein (beta APP) gene of the mouse was disrupted by inserting into exon 2 a cassette containing a neomycin resistance gene and a putative transcription termination sequence. Contrary to expectation, brain and other tissues from mice homozygous for the insertion still contained beta APP-specific RNA, albeit at a level 5- to 10-fold lower than wild type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.

The effect of the size of the tumour and the amount of antibody injected on the biodistribution of a family of radioiodinated antibodies was studied. The intact mouse anti-(carcinoembryonic antigen) (anti-CEA) monoclonal antibody CE-25, its F(ab')2 fragment and the intact human-mouse chimeric from CE 4-8-13 were evaluated in a model system using the human CEA-producing colon xenograft T 380 grown in nude mice. The relative retention (the percentage of the injected dose per gram of tissue), of mouse mAb and F(ab')2 in tumour and most normal tissues 1 day after injection was independent of the antibody dose; after 4 days the mAb values increased with increasing antibody dose. The relative retention of chimeric mAb increased with increasing antibody dose 1 day after injection and also slightly after 4 days. The relative retention in tumour tissue was lower in bigger xenografts for all antibodies. The relative retention of mouse mAb in small tumours increased from day 1 to day 4; for chimeric mAb this value decreased. In normal tissues the relative retention of mouse mAb decreased from day 1 to day 4, but the relative retention of chimeric mAb in normal tissue dropped rapidly and changed little afterwards. Thus the biokinetics of antibodies is "species"-dependent: foreign, mainly human, chimeric antibody clears faster from normal mouse tissue than mouse antibody and reaches lower concentrations.

The following three methods were evaluated in order to obtain a most efficient freezing protocol for the preservation of two-cell mouse embryos: (a) slow cooling and slow thawing in 1.5 M dimethyl sulfoxide, (b) slow cooling and fast thawing in 1.5 M propanediol (PROH), and (c) ultrarapid freezing and fast thawing in either 3.5 M DMSO or 3.0 M PROH. In the slow-cooling procedures (a and b) ice nucleation (seeding) was induced manually or automatically. With method a, only a slight difference, 51.8% for manual and 58.9% for automatic seeding, was observed in survival rates, while the development to blastocysts was significantly affected: 35.4% with manual and less than 10% with automatic induction (P less than 0.001). Method b gave high survival (86.2%) and developmental rates (69.0%) with manual seeding compared with automatic seeding (20.7 and 9.8%, respectively; P less than 0.001). Using protocol c, higher survival and developmental rates were obtained with DMSO (84.8 and 55.9%) than with PROH (39.8 and 19.4%, P less than 0.001). These results demonstrate that inducing nucleation manually is superior to the use of a highly sophisticated autoseeding system and that method b with manual seeding is most effective in preserving the developmental capacity of two-cell mouse embryos after freezing and thawing. There is evidence that this is also true of human embryo cryopreservation.

A nude mouse colony held in an isolation unit was found to harbor MHV despite the fact that all hygienic precautions were taken. The virus spread rapidly causing a high mortality rate predominantly in experimental animals. Moreover, we observed a high percentage of tumor regression in our tumor transplanted mice. Attempts to eliminate the MHV by repeated tumor transplantation into virus-free nude mice were unsuccessful. Since MHV has a limited host range, we transplanted, in parallel, four different lines of embryonic renal tumors (three triphasic nephroblastomas and one malignant rhabdoid tumor of the kidney) from athymic mice into athymic rats and fragments of the same tumors into "fresh" nude mice. All manipulations were performed in isolators. Detection of MHV was done twice by serological examination of six-week-old sentinels. The results showed transmission of MHV infection in the control mice under gnotobiotic conditions as previously found in the normal animal room. On the other hand, there was no evidence of infection, neither in the transplanted nude rats nor after retransplantation of tumors into nude mice. We hypothesize that the virus is harbored in the stromal cells of the murine host but not of the rat host nor in the human tumor cells. Histological comparison showed no alteration of specific tumor morphology in the different hosts.