The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

High-level regulated expression of the human G6PD gene in transgenic mice

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.     

Glutamine: fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats.

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521+/-75 pmol/mg protein x min) in the placenta, 895+/-74 in the liver and 81+/-11 in muscle (p<0.001 between each tissue). In the diabetic rats, GFA activities were approximately 50% decreased both in the liver (340+/-42 pmol/mg protein x min, p<0.05 vs control rats) and in skeletal muscle (46+/-3, p<0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519+/-112 pmol/mg protein x min) and non-diabetic (1521+/-75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.     

Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.     

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.     

Expression of tissue-specific genes in transgenic mice

The ability to introduce cloned genes into mouse germ line has been used for analyzing cis-acting DNA sequences involved in tissue-specific and developmental regulation of the introduced gene. Using this system we have attempted to produce a transgenic mouse model for human dominantly inherited disease, familial amyloidotic polyneuropathy. Recently the mutant transthyretin gene which is considered to be responsible for this disease has been cloned and well characterized at molecular level. We have produced transgenic mice by microinjecting human mutant gene. Amyloid deposition was observed in the mucosa of alimentary tract and renal glomeruli, suggesting that this approach is successful in establishing the mouse model for human genetic disease. In addition, these experiments suggest that the expression of the mutant gene is regulated normally during developmental process and that the cause of adult onset is not due to the dysregulation of this gene expression.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.