Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.     

Inducible gene expression in mammalian cells and transgenic mice

The discovery of the superantigens (SAgs) offered new insights on the interaction between microorganisms and the host immune system. Associated to Major Histocompatibility Complex (MHC) class II molecules, SAgs bind to the variable domain of the beta chain (V beta) of the TCR alpha beta engaged in the family specificity of lymphocytes. Therefore, these molecules are able to activate a high number of T lymphocytes as well as surface MHC class II bearing cells, leading to an overriding release of cytokines and inflammatory mediators, which have been related to their toxic effects. Endogenous SAgs are encoded by murine tumor proviruses (Mtv) which are integrated in the genome of mice. Bacteria and viruses produce exogenous SAgs and those related to food poisoning have been widely studied. The presence of parasite SAgs is still unclear and further studies are required to establish their existence and effects on the corresponding infections.     

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.     

High-level expression of recombinant human fibrinogen in the milk of transgenic mice

Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.     

The myeloperoxidase gene proximal enhancer directs hematopoietic-specific expression in transgenic mice

The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. The MPO gene includes a promoter proximal enhancer which is coincident with DNaseI hypersensitive chromatin sites and is specifically active in myeloid cell lines. We developed transgenic murine lines in which 1.3 kb of murine MPO proximal 5' flanking region DNA was linked to a TATAA homology and RNA initiation site derived from the HSV-TK promoter and to a luciferase reporter (MPOTKLUC). In each of six founder lines, high-level luciferase activity was evident in marrow, thymus and spleen. Modest- to high-level luciferase expression was also evident in brain and in the heart in several of the lines, and luciferase activity was at or near background levels in lung, liver, kidney, stomach, colon, bladder, skeletal muscle, skin and small intestine in all of the MPOTKLUC transgenic mice. Within marrow cells, luciferase activity was evident in myeloid (GR-1+), B lymphoid (B220+) and T-lymphoid (CD4+) cells. Additional regulatory regions, thus, may be required to further restrict MPO gene expression to immature myeloid cells.     

Characterization of human endotoxin lipopolysaccharide receptor CD14 expression in transgenic mice

CD14 is a major receptor for the bacterial endotoxin LPS. Since CD14 is specifically and highly expressed on the surface of monocytic cells, it has been used as a monocyte/macrophage differentiation marker. To identify elements that are critical for the direction of the tissue-specific expression of CD14, an 80-kb genomic DNA fragment containing the coding region of the CD14 gene, as well as a considerable amount of both upstream and downstream sequence, was used to generate transgenic mice. The analysis of mice from six different founder lines demonstrated that this genomic DNA fragment was sufficient to direct human CD14 gene expression in a monocyte-specific manner among hematopoietic cells. Furthermore, the data lead us to a new finding that CD14 is highly expressed in the human liver, a primary organ involved in the acute phase response. These transgenic mice provide a useful model to analyze the biological function of human CD14.     

Tissue-specific regulation of angiotensinogen gene expression in spontaneously hypertensive rats

Angiotensinogen is expressed in many tissues besides the liver. Recent studies have suggested that abnormalities in the regulation of angiotensinogen gene expression may be involved in the development of hypertension. However, little information is available concerning the functional significance of tissue angiotensinogen. In this study, we measured plasma angiotensinogen concentration by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Although plasma angiotensinogen concentration in SHR was comparable to that in WKY at 6 weeks of age, it was increased significantly at 14 weeks of age in SHR and became higher than that in WKY. The levels of hepatic angiotensinogen mRNA were similar in SHR and WKY, and the levels of aortic, adrenal, and renal angiotensinogen mRNAs were lower in SHR than in WKY at both 6 and 14 weeks of age. Brain angiotensinogen expression in SHR was higher than in WKY at 6 weeks of age and was comparable to that in WKY at 14 weeks of age. On the other hand, cardiac and fat angiotensinogen mRNA levels were significantly increased at 14 weeks of age in SHR. These results demonstrate that the expression of tissue angiotensinogen is regulated differently in SHR and WKY and indicate that the development of hypertension is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as cardiac and adipogenic angiotensinogen mRNA in SHR.     

High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice

We studied the development of liver tumors in male HBx gene transgenic mice. Of two lineages studied, in the lineage with the lowest HBx gene expression liver tumors developed only in an incidence comparable with that in normal CD-1 strain, whereas 84% of male mice with a high level of the HBx gene product succumbed to liver neoplasia, indicating that continued HBx gene expression higher than a certain threshold level may be necessary for the development of hepatic neoplasia. Sixty-five mice from a lineage with a high level of HBx expression were then followed throughout their 24-mo lifespan. The livers of transgenic mice showed foci of cellular alteration with cytoplasmic vacuolations around the central veins from the age of 2 mo, but these foci did not expand progressively by the age of 12 mo. Immunostaining demonstrated such hepatocytes had higher expression of HBx protein than surrounding cells. Neoplastic lesions including liver cell adenomas and hepatocellular carcinomas developed from the age of 13 mo. By bromodeoxyuridine labeling analysis, hepatocytes in altered foci were found to have increased DNA synthesis, whereas no labeling was observed in age- and sex-matched nontransgenic littermate controls. Furthermore, DNA content analysis revealed the existence of several small aneuploid peaks in the transgenic liver before the age of tumor development. These results suggest that the continued expression of HBx gene may initiate a complex process to hepatocellular carcinoma by inducing DNA synthesis and placing large numbers of hepatocytes subjective to secondary events for transformation.     

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.     

Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice

Human erythropoietin (Epo) gene expression is inducible by hypoxia or anaemia in the kidney and liver. Previous transgenic mouse experiments have demonstrated that sequences required for Epo gene induction in the kidney reside in a 7 8 kb Barn HI fragment located 6 kb upstream of the gene. To sublocalize these sequences, we performed Desoxyribonuclease I (DNAse I) mapping studies using transgenic mice which carried this DNA fragment. These studies revealed a DNAse I hypersensitive site (DNAse I HS) located 4 6 kb from the upstream end of the 7.8 kb fragment in anaemic kidney and liver samples. Sequence analysis of the region encompassing the DNAse I HS revealed an element with remarkable homology to the 3' Epo gene hypoxia-inducible enhancer. This suggested the presence of an additional regulatory element that contributes to the control of hypoxia-inducible Epo gene expression in kidney and liver. We constructed transgenic mice containing the human Epo gene linked to either the 5 kb upstream or 2.5 kb downstream portion of the 7.8kb fragment. Inducible expression was limited to the liver. Thus, neither fragment was alone sufficient to confer kidney inducible expression. These findings indicate that sequences more than 8.5 kb upstream of the Epo gene are required for kidney-specific induction. They suggest that either those sequences reside in an 0.3 kb Hind III fragment located between the 5 kb and the 2.5 kb fragments or that sequences in the 5 kb or 0.3 kb fragments must interact with sequences in the 2.5 kb fragment to allow Epo gene induction in the kidney.     

A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice

Expression of transgenes in mice often fails to follow the normal temporal and spatial pattern and to reach the same level as the endogenous copies. Only in exceptional cases has position-independent and copy number-dependent expression been reproduced. The size constraint of standard constructs may prevent the inclusion of important remote regulatory elements. Yeast artificial chromosomes (YACs) provide a means of cloning large DNA fragments and the transfer of YAC DNA into somatic cells has been reported. We have previously produced transgenic mice carrying a 35 kilobase YAC construct. Here we report the transfer of a 250 kilobase YAC covering the mouse tyrosinase gene into mice by pronuclear injection of gel-purified YAC DNA. The YAC was inserted into the mouse genome without major rearrangements and expression of the YAC-borne tyrosinase gene resulted in complete rescue of the albino phenotype of the recipient mice. Expression from the transgene reached levels comparable to that of the endogenous gene and showed copy number dependence and position independence.     

Analysis of the human ferrochelatase promoter in transgenic mice

Ferrochelatase catalyzes the chelation of ferrous iron and protoporphyrin to form heme. It is expressed as a housekeeping gene in all cells, but is upregulated during erythropoiesis. Ferrochelatase activity is deficient in the inherited disease protoporphyria as a result of heterogeneous mutations. Although human ferrochelatase is transcribed from a single promoter in both nonerythroid and erythroid cells, previous studies using transient transfection assays failed to demonstrate erythroid-specific increased expression from 4.0 kb of the human ferrochelatase promoter containing the erythroid cis-elements, GATA and NF-E2. The present study analyzes the in vivo regulation of the ferrochelatase gene to provide insights into the mechanism of its erythroid-specific enhancement. Transgenic (TG) mouse lines were generated in which the luciferase reporter gene was driven by either a 150-bp ferrochelatase minimal promoter (-0.15 TG) or by a 4.0 kb extended 5' upstream region (-4.0 TG). Expression of the -4.0 TG transgene was generally consistent with the endogenous gene during embryonic development and in nonerythroid and erythroid tissues as demonstrated by Northern blotting and mRNA in situ hybridization. The -4.0 TG was expressed at a higher level than the -0.15 TG in nonerythroid and erythroid tissues, including during extramedullary erythropoiesis induced by n-acetylphenylhydrazine injection. The enhanced erythroid expression of the -4.0 TG correlates with the appearance of a DNase I hypersensitive site in the 5' flanking region of the transgene. Therefore, in the context of chromosomal integration, the 5' flanking region of the ferrochelatase gene is necessary and sufficient to confer high levels of transgene expression in erythroid tissue.     

Expression of the rat arginine vasopressin gene in transgenic mice

A line of mice has been developed which are transgenic for an 8.2-kilobase (kb) genomic clone of the rat vasopressin (VP) gene. Using a polymerase chain reaction technique, the rat VP (rVP) transgene was shown to have tissue-specific mRNA expression in the hypothalamus, temporal lobe, parietal cerebral cortex, cerebellum, and posterior pituitary, similar to the tissue distribution of endogenous mouse and rat VP expression. Expression of transgenic rVP mRNA was also found in the lung and pancreas of the transgenic mice, sites of known ectopic expression of VP. Using two methods, Northern blot analysis with species-specific cRNA probes and a quantitative polymerase chain reaction technique, the quantity of rVP transgene mRNA was shown to regulate appropriately in response to an osmotic stimulus. After 72 h of water deprivation, the quantity of transgenic rVP mRNA increased 6.8 +/- 3.0-fold. This was not significantly different than the fold increase in mouse VP mRNA quantity seen in nontransgenic mice (4.8 +/- 1.5) but was significantly different (P < 0.05) than the 1.2 +/- 0.03-fold increase in rat VP mRNA seen in normal rats after water deprivation. In the rat hypothalamus, VP mRNA poly(A) tail length increases with osmotic stimulation, while in the mouse it does not. The poly(A) tail of transgenic rVP mRNA expressed in mouse hypothalamus did not increase in length after osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human gamma-interferon expression in the mammary gland of transgenic mice

Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.