Nephrogenic diabetes insipidus in an Australian aboriginal kindred.

Four Australian aboriginal children were found to have nephrogenic diabetes insipidus. They are the sons of 3 sisters who were shown to have a urinary concentrating defect uncorrected by vasopressin. An extensive genealogy did not reveal any caucasian genetic influence suggesting that a new genetic mutation was responsible.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

Murine peripherin gene sequences direct Cre recombinase expression to peripheral neurons in transgenic mice

Group B streptococcus (GBS) induced macrophage apoptosis by which it could avoid host defence mechanisms. Macrophages, which constitutively express phosphatidylserine (PtdSer) on the outer leaflet of plasma membrane, increased PtdSer exposure during GBS-induced apoptosis. Induction of apoptosis decreased PtdSer radioactivity of macrophages incubated with [³H]serine. The effect appeared not due to increasing conversion of PtdSer to phosphatidylethanolamine or phosphatidylcholine nor to the release of radioactive membrane vesicles. The radioactivity in lysoPtdSer was also reduced. These results confirm that induction of apoptosis involves a modification of PtdSer metabolism and point out the typical features of the GBS-induced apoptosis with respect to other models of apoptosis.     

Neurofilament gene expression in transgenic mice

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.

Dopamine beta-hydroxylase (DBH) catalyzes the final step in the biosynthesis of norepinephrine, the principal classic neurotransmitter of peripheral sympathetic neurons. We have shown that 5.8 kb of 5' upstream region from a cloned human DBH gene promoter is sufficient to direct expression of the E. coli lacZ gene in transgenic mice to neurons of the locus ceruleus and other classic noradrenergic brain stem nuclei, sympathetic ganglion neurons, and adrenal chromaffin cells. lacZ expression was also observed in neurons of the enteric system, the retina, some sensory and all cranial parasympathetic ganglia, and some diencephalic and telencephalic brain nuclei. The expression pattern of the transgene in DBH-immunonegative sites overlapped with many sites where expression of tyrosine hydroxylase or phenylethanolamine N-methyltransferase, two other catecholamine biosynthetic enzymes, has been reported.     

Introduction and expression of the human Bs-globin gene in transgenic mice.

Owing to the episodic and unpredictable nature of the sickling crisis, many aspects of the disease sickle cell anemia have resisted in vivo analysis. The lack of an animal model has hindered the pathophysiological investigation of this disease, as well as deterred the development of pharmacological therapies. The transgenic mouse system offers a new means for creating animals that make a specified mutant gene product, and we have used this system to create a series of mice that contain the human beta s-globin gene. These animals express this gene in the appropriate tissues and at the same point in development as the adult mouse globin genes are expressed. We have crossed the human beta s-containing transgenic mice with a beta-thalassemic mouse line and examined the hemoglobins produced by these mice. Their red cells contain 10% mouse alpha/human beta s hybrid hemoglobin, which partially corrects the thalassemic phenotype of the homozygous beta-thalassemic animals. Though the red cells do not sickle, other properties of the human beta s gene in these mice indicate the potential for the eventual development of a transgenic animal model for sickle cell anemia.     

Cloning of the goat beta-casein-encoding gene and expression in transgenic mice.

The goat beta-casein-encoding gene (CSN2), which encodes the most abundant protein of goat milk, has been cloned and sequenced. The intron/exon organization of the 9.0-kb goat CSN2 gene is similar to that of other CSN2 genes. Expression of the goat gene was principally restricted to the mammary gland of lactating transgenic animals. A low level of expression was also observed in skeletal muscle and skin. In contrast to a rat CSN2 transgene [Lee et al., Nucleic Acids Res. 16 (1988) 1027-1041], the goat gene was expressed to a high degree in the lactating mammary gland. Differences in the content or context of regulatory elements may account for the enhanced performance of the goat relative to the rat CSN2 gene in transgenic mice.     

Inducible gene manipulations in brain serotonergic neurons of transgenic rats

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.     

Tissue-specific expression of theHLA-DRA gene in transgenic mice

Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class IIHLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon- (IFN)- induced] of the human products in most mouse tissues. The tissue distribution of theDRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution ofDRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of theDRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greately reducedDRA levels in spite of a normal expression of the endogenous Ea molecules.     

Tissue-specific expression of a vimentin--desmin hybrid gene in transgenic mice.

We have introduced a hybrid gene, pVVim2, composed of the 5' region of the hamster vimentin gene encoding the head and rod domain of vimentin and the 3' region of the hamster desmin gene encoding the tail domain of desmin, into the germ line of mice by pronuclear injection. RNA and protein analysis of mice transgenic for this construct showed that the pVVim2 gene was expressed at high levels in a developmental and tissue-specific manner. This indicates that the vimentin-derived segment of the fusion gene contains all the regulatory elements required for vimentin-specific expression. Immunohistochemical staining of fibroblast cultures derived from the transgenic mice with antibodies specific for vimentin and desmin demonstrated that the pVVim2 protein is assembled into filaments that co-localize with the endogenous vimentin filaments. The expression of pVVim2 protein in mesenchymal cells does not interfere with the function of vimentin in these cells.     

Vasopressin and ethanol preference. II. Altered preference in two strains of diabetes insipidus rats and nephrogenic diabetes insipidus mice

In the first paper of this series, the influence of a single gene (di) for vasopressin deficiency on ethanol intake in rats was demonstrated. We studied preference for concentrations of ethanol between 2.2 and 10 percent versus tap water in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Treatment with vasopressin or related peptides restored ethanol drinking to normal but also corrected water balance. In the experiments reported here, Roman High Avoidance (RHA) rats of three genotypes (+/+, di/+, and di/di) were also tested for ethanol intake and preference with similar but not identical results. Thus, the effects of the di gene are independent of the genetic background on which it is placed to at least some extent. Chlorothiazide, a drug unrelated to vasopressin, also normalized ethanol drinking and corrected water balance in di/di rats. In nephrogenic diabetes insipidus mice, there was a strong negative correlation between severity of polydipsia and preference for ethanol. Thus, no paradigm tested was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state.     

Germ cell-specific expression of a proacrosin-CAT fusion gene in transgenic mouse testis.

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.