Rat synapsin 1 promoter mediated transgene expression in testicular cell types.

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.

Spontaneous hepatocellular carcinoma is reduced in transgenic mice overexpressing human O6- methylguanine-DNA methyltransferase.

O(6)-methylguanine (O(6)mG) is a potent mutagenic and procarcinogenic DNA lesion. Organisms have evolved with a DNA repair mechanism that largely ameliorates the deleterious effects of O(6)mG through a direct reversal mechanism by a protein termed O(6)-methylguanine-DNA methyltransferase (MGMT). However, the contribution of O(6)mG to carcinogenesis, in the absence of known exposure to agents that produce it, has not been defined. Nontransgenic C3HeB male mice have a high frequency of spontaneous liver tumors. Transgenic CeHeB/FeJ mice expressing human MGMT (hMGMT) were generated that had elevated hepatic MGMT activity. The spontaneous development of hepatocellular carcinoma was significantly reduced in those mice expressing hMGMT compared with nontransgenic C3HeB/FeJ male mice. No differences were detected in spontaneous mutant frequencies in lacI transgenes in mice carrying hMGMT compared with that without hMGMT but the proportion of GC to AT transition mutations was lower in the transgenic mice carrying hMGMT as well as lacI. Tumors that arose in C3HeB/FeJ transgenic mice were largely deficient in hMGMT protein as determined by immunohistochemistry with a monoclonal antibody directed against hMGMT. Together these data indicate that spontaneous O(6)mG lesions induced hepatocellular carcinogenesis in C3HeB/FeJ male mice. These transgenic mice represent a rare example of reduced spontaneous carcinogenesis.

Gap junctional communication between the satellite cells of rat dorsal root ganglia.

Many studies have described the ultrastructure of the dorsal root ganglia in various embryonic and adult animals, but in spite of the efforts of many investigators the functional role of the satellite cells in this tissue is not clearly understood. In this study, we discuss the function of this cell type based on the concept of cell-to-cell interaction through gap junctions. Five male 60 day-old Wistar strain rats were used. All animals were anesthetized with pentobarbital and perfused with glutaraldehyde fixative, then the dorsal root ganglia in levels L4, L5 and L6 were taken from each rat. After postosmication, the specimens were prepared for observation by transmission electron microscopy. All nerve cells were completely surrounded by satellite cell cytoplasmic expansions. The boundaries between adjacent nerve cells and satellite cells were complicated due to the presence of perikaryal projections of nerve cells. Gap junctions which showed the typical trilamellar structure of plasma membranes were found mainly between satellite cell processes belonging to the same nerve cell. On the other hand, some gap junctions were found between the satellite cell projections belonging to different nerve cells. The size of the gap junctions ranged from 300 to 400 nm. No gap junctions were associated with the plasma membrane of any nerve cell. In conclusion, only satellite cells can share free transcellular exchange of cytoplasmic molecules such as ions, amino acids, sugars and several second messengers including cAMP and inositol 1,4,5-triphosphate by way of gap junctions in dorsal root ganglia.

Effects of hydrocortisone on the formation of gap junctions and the abnormal growth of cilia within the rat anterior pituitary gland: possible role of gap junctions on the regulation of cell development.

We investigated the effects of hydrocortisone on the formation of gap junctions in and the growth of cilia on folliculo-stellate cells. The male rats of experimental groups were given daily intraperitoneal injections of 5 mg/kg of hydrocortisone from Day 20 to 60. Five rats were killed at ages 10, 20, 30 and 40 days after initiation of injections, and the pituitary gland was removed from each rat. Then, the specimens were prepared for observation by transmission electron microscopy. A delay in the formation of gap junctions between folliculo-stellate cells was observed in hydrocortisone treated rats compared with control rats on Day 30, 40 and 50. Another finding in the present study was the increase of ciliated follicles on Day 40 and 50 in the hydrocortisone treated groups, simultaneous with the delay in gap junction formation. The results suggest that hydrocortisone has a suppressive effect on the gap junction formation between folliculo-stellate cells, and loss of intercellular communication by way of gap junctions may lead to alteration of morphological development of the cell.

Health span and life span in transgenic mice with modulated DNA repair.

One way to better understand the contribution of DNA repair, DNA damage, and mutagenesis in aging would be to enhance DNA repair activity, lower DNA damage, and lower mutagenesis. Because the repair protein O6-methylguanine-DNA methyltransferase (MGMT) acts alone and stoichiometrically, the human MGMT (hMGMT) cDNA was selected to test the feasibility of enhancing DNA repair activity in transgenic mice. MGMT activity is largely responsible for ameliorating the deleterious effects of O6-methylguanine (O6mG) lesions in DNA in a direct reversal mechanism. A transgene was constructed consisting of a portion of the human transferrin (TF) promoter and hMGMT cDNA such that hMGMT is expressed in transgenic mouse brain and liver. Expression of hMGMT was associated with a significant reduction in the occurrence of an age-related hepatocellular carcinoma in male mice at 15 months of age. Longitudinal and cross-sectional studies were initiated to determine whether the reduced incidence of hepatocellular carcinoma would impact median or maximum life span. The cross-sectional study performed on 15-month-old male animals confirmed the reduced occurrence of spontaneous hepatocellular carcinoma. At 30 months of age, however, the occurrence of hepatocellular carcinoma in at least one transgenic line was similar to that for nontransgenic animals. The longitudinal study is ongoing; however, at present no significant differences in life span have been detected. Tissues expressing the MGMT transgene also displayed greater resistance to alkylation-induced tumor formation. These results suggest that transgenes can be used to direct enhanced DNA repair gene expression and that enhanced expression can protect animals from certain spontaneous and induced tumors.

Pulmonary expression of the human haptoglobin gene.

Haptoglobin (Hp), a member of the acute-phase reactants, has long been known as a major hemoglobin-binding protein associated with hemoglobin catabolism. Recent studies indicate that another important biologic function of Hp is the modulation of the immune response. We found that Hp is expressed at high levels in specific cells, including alveolar macrophages and eosinophils in diseased or inflamed human lung tissues, but not in the normal lung. Expression of the human Hp gene was studied in two transgenic mouse lines carrying a 9-kb human Hp 2 gene. In both lines, the human Hp transgene was expressed constitutively in alveolar macrophages at a high level, whereas the endogenous mouse Hp was synthesized in airway epithelial cells. Expression of the human Hp transgene in lung cells was upregulated when the transgenic mice were treated with endotoxin. In humans and in Hp transgenic mice, human Hp messenger RNA was also detected in circulating eosinophils, but not in other blood cells. Our findings suggest that Hp is involved in a variety of lung inflammatory diseases, including respiratory allergy and asthma. The transgenic mouse line that overexpresses the human Hp gene in alveolar macrophages and eosinophils is a promising system for investigating the function of Hp in vivo during lung inflammation.

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase).

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.

Effects of a low-protein diet on prolactin- and growth hormone-producing cells in the rat pituitary gland.

BACKGROUND: It is well known that an unbalanced diet induces various changes in the pituitary gland. However, little attention has been paid to the molecular aspects of this perturbation. We studied the influence of a low-protein diet (LPD) on the prolactin (PRL) and growth hormone (GH) cells in the rat pituitary gland using immunohistochemical staining and in situ hybridization. MATERIALS: Rats aged 20 days were fed a diet containing 27% protein or one with 8% protein (LPD) for 30 days. Pituitary glands were obtained and subjected to either immunohistochemistry or in situ hybridization. Quantitative morphological analysis was then conducted to determine cell number and area as well as the percentage of cells stained by the respective antisera and/or cDNA probe in each experimental group. RESULTS: The average sectional areas of both PRL- and GH-producing cells in the LPD group were smaller in size than those in the controls. The cell numbers per unit area (mm2) of PRL-positive cells and PRL mRNA-positive cells were 3,596.5 and 3,948.6, respectively, in the LPD group, and 3,179.6 and 4,888.5, respectively, in the controls. The numbers per unit area of GH-positive cells and GH mRNA-positive cells in the LPD group were similar (2,252.3 and 2,224.4), as compared to 2,161.3 and 1,684.2, respectively, in the well-fed rats. Whereas PRL-positive cells comprised about 27% of the total number of cells in both animal groups, those given the LPD contained a lower percentage (29%) of PRL mRNA-positive cells as compared to the controls (44%). On the other hand, GH mRNA-positive cells numbered about 15% of the total cell population both animal groups; however, the malnourished rats contained a lower percentage (16%) of GH-positive cells than did their well-fed counterparts (20%). CONCLUSIONS: Taken together, these results indicate that in the rat pituitary gland, administration of an LPD reduced the size of PRL- and GH-positive cells as well as differentially affecting a subpopulation of the PRL mRNA-positive cells and the GH-positive cells.

Biogenesis and function of lipolysosomes in developing chick hepatocytes.

Proliferation of lipolysosomes is one of the characteristic aspects of embryonic chick hepatocytes. Formation of lipolysosomes is observed in the well-developed trans-Golgi network, with the highest frequency occurring from 11 to 14 days of incubation. The lipolysosomes usually contain a small and electron-dense lipid inclusion; however, during development, they gradually enlarge with an accompanying reduction in the electron density of the inclusion. Lipolysosomes isolated from neonatal chick liver homogenates were mainly composed of esterified cholesterol and showed considerably high activity of lysosomal enzymes. Moreover, the lipolysosome fraction is clearly shown to be a function of intralysosomal lipolysis via acid lipase. This accumulation of esterified cholesterol within lipolysosomes might be attributed to an excessive uptake and conversion of plasma lipoproteins to lipolysosomes. This concept is supported by the appearance of an abundance of coated pits and both "early" and "late" endosomes. The major components of plasma lipoprotein are low density lipoprotein (LDL) and high density lipoprotein (HDL), the cholesterol-rich lipoproteins, whose cholesterol content increases during the last week of incubation when the lipolysosomes quickly enlarge. Plasma lipoprotein particles are produced in the yolk sac epithelium from yolk very low density lipoprotein (VLDL) and transferred via the vitelline circulation to the chick liver. After hatching, when the supply of nutrients from the yolk sac is terminated, lipolysosomes immediately decrease in size and number. The cholesterol and fatty acids released are useful as an energy source and lipid metabolism in general, especially after hatching. Food intake induces the use of and accelerates the disappearance of lipolysosomes. Instead of lipolysosomes, lipid droplets appear and increase in number and size with concomitant increases of triglyceride concentrations in the liver homogenates, suggesting that lipogenesis has begun in the chick hepatocyte.

Folliculo-stellate cells and intercellular communication within the rat anterior pituitary gland.

Folliculo-stellate (FS) cell are agranular and arranged around a follicle. They contain the S-100 protein and beta-adrenergic receptors. It has been suggested that they can act as stem cells, since they show mitotic figures, and could transform into granular or chromophilic cells according to the concept of a "cell renewal system." Cell-to-cell interactions among pituitary cells have been described, and recent progress with freeze-fracture electron microscopy has provided novel observations of the cell surface and gap junctions within the rat or teleost fish pituitary gland, or in cultured rat pituitary cells. In adult rats, the anterior pituitary was composed of lobules incompletely separated by a basement membrane. Follicles consisted exclusively of FS cells. Gap junctions were observed only between adjacent FS cells, in rare cases on the tips of their cytoplasmic processes. Thus, the FS cells, connected by gap junctions, made up a dense cellular network throughout the pituitary. Gap and tight junctions were absent on granular cells. Elongated follicles with columnar FS cells were observed in 10-day-old rats and were separated into smaller units. The number of gap junctions rapidly increased with age until 40-45 days of age. Few S-100 protein positive cells were observed on day 10, along the marginal cell layer and near the so-called postero-lateral wing. The frequency of positive cells increased with age and by day 40; numerous cells were observed throughout the anterior lobe. Gap junction number also varied with the stage of the estrous cycle, and frequency; during diestrus, they were half of that during proestrus or estrus. The number of gap junctions increased in late pregnancy and in lactating rats, probably due to changes in estrogen and progesterone. Hormone (LH-RH and testosterone) treated groups of rats showed accelerated development by almost 10 days, compared with controls. In castrated male rats, the ultrastructure of the pituitary remained immature even at 40 days of age, when the number of gap junctions was a quarter or less than the number in intact rats. Testosterone treatment restored the frequency of gap junctions to a normal level. We conclude that the appearance of gap junctions in the pituitary cells and maturation of the gland are dependent to a large degree upon gonadal steroids.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide.

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

Sealing of the follicular lumen of the anterior pituitary gland of the male rat.

It is commonly accepted that follicular lumina of the adult rat anterior pituitary gland are tightly sealed by junctional complexes, especially tight junctions. In this report, we describe the presence of follicular lumina that are unsealed. Peroxidase (HRP) was used to study such structures and when injected through the femoral vein, was observed in association with a few follicular lumina, on their microvilli and around the cilia of folliculo-stellate cells. The existence of peroxidase-positive follicles clearly shows that follicles of the hypophysis are not always firmly sealed by tight junctions. The folliculo-stellate cells which faced the peroxidase-positive follicles displayed HRP deposits which were membrane bound within their cytoplasm. These findings suggest an absorptive function for the folliculo-stellate cells.

Participation of endodermal epithelial cells on the synthesis of plasma LDL and HDL in the chick yolk sac.

We ultrastructually examined the chick yolk sac endodermal epithelium and evaluated our findings in combination with the biochemical analysis of serum and yolk lipoproteins. Twenty-five to 30 nm-sized particles were demonstrated to be a principal element of the extracellular yolk mass and these were determined to be yolk very low density lipoprotein (VLDL). The particles were shown to be taken up by the epithelial cells via coated pits and engulfed by plasma membrane invaginations together with yolk subdroplets, another element of the yolk mass. Through apical vacuoles, the two yolk elements were incorporated into yolk drops, which were identified to be one of the lysosomal structures by a cytochemical procedure using acid phosphatase (AcP)ase activity. During the last week of incubation, which is the final third of the incubation period, the digestion seemed to progress rapidly in the yolk drops, which came to resemble lipolysosomes; lipoprotein production became active as expressed by an enlarged Golgi apparatus. The newly produced lipoprotein particles were electron-lucent and irregular in size (50-120 nm). They were sequestered in secretory vacuoles and secreted from the vascular surface of the epithelial cells. Finally, the particles were thought to be taken into the vitelline circulation as plasma lipoproteins. The major component of lipoprotein in serum was determined to be low density lipoprotein (LDL) and high density lipoprotein (HDL), while cholesterol content was found to increase during incubation. We concluded that endodermal epithelial cells participate the synthesis of plasma LDL and HDL. For this synthesis the cells probably apply lipids and apo-protein generated from yolk VLDL degradation.

Mg(2+)-dependent and Ca2+, Mg(2+)-dependent ATPase activities in the harderian gland of rodents: age and sex influences.

Three experiments employing male and female Syrian hamsters (aged 1, 2, and 8-10 months), male Sprague-Dawley rats (aged 1, 2, and 10 months) and male C57B1 mice (aged 2, 7, 13, and 29 months) examined the effects of age and sex on Mg(2+)-dependent and Ca2+, Mg(2+)-dependent ATPase activity in the Harderian gland. Significant differences due to age and sex were observed in the hamsters and rats but not with age in mice. Generally, male hamsters had significantly higher Mg(2+)-dependent and Ca2+, Mg(2+)-dependent (exception at one timepoint) ATPase activity than did females. Age-matched male and female rats had similar values of Mg(2+)-dependent ATPase activity, but males had significantly higher Ca2+, Mg(2+)-dependent ATPase activity than females at 2 months of age.

In vivo uptake of lecithin-coated polystyrene beads by rat hepatocytes and sinusoidal endothelial cells.

BACKGROUND: While phagocytosis by Kupffer cells (stellate perisinusoidal macrophages) is well known and that by endothelial cells also is thought to occur under certain conditions, the uptake of large particles by hepatocytes has not been well studied. We reported previously the selective phagocytic uptake of material by hepatocytes using egg lecithin-coated silicon particles. In the present work, we describe more precisely this process following the injection of lecithin-coated polystyrene beads. Additionally, we consider the possible significance of the transcytotic action by endothelial cells. METHODS: Polystyrene latex beads (240 nm in diameter) composed of two layers of polystyrene and methyl methacrylate with a central void cavity and diameter of 140 nm were injected into male Wister-Imamichi rats. The injections were administered through the hepatic portal vein in a volume of 3 ml (concentration of the lecithin-coated or uncoated beads was 2 mg/ml). Controls received the lecithin alone at a concentration of 2 mg/ml. Liver samples were taken 5, 10, or 15 min after injection, fixed, and processed for ultrastructural analysis. RESULTS: Both lecithin-coated and noncoated beads were mainly incorporated in the Kupffer cells as well as in the endothelial cells. Bristle-coated invaginations were observed in the uptake by both cell types; however, noncoated invaginations were also active in the endothelial cells, especially on the surface facing the perisinusoidal space of Disse. Only coated beads were observed within the space or in the hepatocytes. Once taken up by the hepatocytes, the lecithin-coated beads were found either within lysosomes or in a free state in the cytoplasm. CONCLUSIONS: Uptake of 240 nm lecithin-coated polystyrene beads was observed by Kupffer cells, endothelial cells and hepatocytes. These beads were considered to be transported across the endothelial cells by transcytosis. Pseudopodia and bristle-coated invaginations were not employed by the hepatocytes when incorporating the beads.

Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation.

Ceruloplasmin (CP) is an important extracellular antioxidant and free radical scavenger. Although CP is expressed mainly in the liver, recent studies have identified the lung as another major site of CP synthesis. The sites and cell types that are responsible for CP expression in baboon and mouse lung are described. CP mRNA is detected in primordial bronchial epithelium in baboon fetuses by 60 days of gestation. At 140 days of gestation and thereafter, CP mRNA is found in airway epithelium and in the ductal cells of the submucosal glands. In developing and mature mice, CP mRNA is present in epithelial cells throughout the airway. In endotoxin-treated mice, the amount of CP mRNA increases several-fold in large airways but increases only moderately in small airways. This suggests that the high concentration of CP in the mucus lining of the upper airway, which serves to filter harmful substances, is particularly important during stressful conditions. Endotoxin treatment in mice also results in the induction of high levels of CP mRNA in a subset of alveolar wall cells. The data suggest that the airway epithelial cells are the major source of CP in the lung fluid and support ceruloplasmin's critical role in host defense against oxidative damage and infection in the lung.

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.

YY1 and Sp1 transcription factors bind the human transferrin gene in an age-related manner.

The iron-binding protein transferrin has major roles in transporting, delivering, and sequestering ferric ions acquired by body tissues. Yet, during aging, serum transferrin levels decrease in humans. Likewise, in transgenic mice carrying chimeric human transferrin transgenes, liver expression of transferrin transgenes decreases with age. The aging regulation is due to decreased gene transcription. Electrophoretic mobility shift assays and antibody-recognition have revealed the binding of 5' regulatory elements of the human transferrin gene by three YY1 proteins, called YY1, YY1-a, and YY1-b, and an Sp1-a transcription factor. An age-related increase in YY1-a and YY1-b binding activities and a decrease in Sp1-like binding activity were shown. Since Sp1 is a positive transcription factor and YY1 can be a negative transcription factor, the alterations in their binding with age could cause the decreased transcription of the human transferrin transgene, and also the age-related decreased serum transferrin levels in humans.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.