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PubMed2004

Insulin-like growth factor-binding protein-4 inhibits growth of the thymus in transgenic mice.

Numerous in vitro studies have demonstrated that IGF-binding protein (IGFBP)-4 is a consistent inhibitor of IGF actions. In order to investigate the functions of IGFBP-4 in vivo, transgenic mice were generated by microinjection of a transgene, in which the murine Igfbp4 cDNA is driven by the H-2K(b) promoter, and followed by a splicing cassette and polyadenylation signal of the human beta-globin gene. Transgene mRNA was expressed ubiquitously, and elevated IGFBP-4 protein was detected in the spleen, thymus, kidney and lung of transgenic mice. The activities of serum IGFBPs were not changed in transgenic mice. Immunohistochemical studies revealed transgene expression predominantly in the thymic medulla and red pulp of the spleen. Body weight and the weights of the spleen, kidney and lung of transgenic mice were not different from controls. In contrast, the thymus of transgenic mice showed a significantly reduced weight and cortex volume. In transgenic thymus and spleen, cell proliferation was inhibited and apoptosis was stimulated. Transgenic mice showed normal T- and B-cell development and normal basal plasma immunoglobulin levels. In conclusion, overexpression of IGFBP-4 inhibits growth of the thymus. IGFBP-4 excess inhibits cell proliferation and stimulates apoptosis in lymphoid tissues, but does not affect lymphocyte development. These findings suggest that IGFBP-4 is a potential growth inhibitor of lymphoid tissues.

Animals↗
PubMed2001

Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice.

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.

Animals↗
PubMed1999

Characterization of a model of hydrocephalus in transgenic mice.

OBJECT: The purpose of this study was to elucidate the pathophysiological characteristics of hydrocephalus in a new transgenic model of mice created to overproduce the cytokine transforming growth factor-beta1 (TGFbeta1) in the central nervous system (CNS). METHODS: Galbreath and colleagues generated transgenic mice that overexpressed TGFbeta1 in the CNS in an effort to examine the role of this cytokine in the response of astrocytes to injury. Unexpectedly, the animals developed severe hydrocephalus and died. The authors have perpetuated this transgenic colony to serve as a model of congenital hydrocephalus, breeding asymptomatic carrier males that are heterozygous for the transgene with wild-type females. One hundred twelve (49.6%) of 226 mice developed clinical manifestations of hydrocephalus, characterized by dorsal doming of the calvaria, spasticity, limb tremors, ataxia, and, ultimately, death. The presence of the TGFbeta1 transgene was determined by performing polymerase chain reaction (PCR) analysis of sample tail slices. Animals with the hydrocephalic phenotype consistently carried the transgene, although some animals with the transgene did not develop hydrocephalus. Animals without the transgene did not develop hydrocephalus. Alterations in brain structure were characterized using magnetic resonance (MR) imaging, gross and light microscopic analysis, and immunocytochemical studies. Magnetic resonance imaging readily distinguished hydrocephalic animals from nonhydrocephalic controls and demonstrated an obstruction at the outlets of the fourth ventricle. Gross and light microscopic examination confirmed the MR findings. The results of immunofluorescent staining of brain tissue slices revealed the presence of the TGFbeta1 cytokine and its receptor preferentially in the meninges and subarachnoid space in both hydrocephalic and control mice. Reverse transcriptase-PCR analysis demonstrated tissue-specific expression of the TGFbeta1, gene in the brains of transgenic mice, and enzyme-linked immunosorbent assay confirmed overexpression of the TGFbeta1 cytokine in brain, cerebrospinal fluid, and plasma. CONCLUSIONS: The transgenic murine model provides a reproducible representation of congenital hydrocephalus. The authors hypothesize that overexpression of TGFbeta1 in the CNS causes hydrocephalus by altering the environment of the extracellular matrix and interfering with the circulation of cerebrospinal fluid. A model of hydrocephalus in which the genetic basis is known should be useful for evaluating hypotheses regarding the pathogenesis of this disorder and should also help in the search for new treatment strategies.

Animals↗
PubMed2000

Aging increased amyloid peptide and caused amyloid plaques in brain of old APP/V717I transgenic mice by a different mechanism than mutant presenilin1.

Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V717I) (Moechars et al., 1999a) was now demonstrated not to affect the normalized levels of alpha- or beta-cleaved secreted APP nor of the beta-C-terminal stubs. This indicated that aging did not markedly disturb either alpha- or beta-secretase cleavage of APP and failed to explain the origin of the massive amounts of amyloid peptides Abeta40 and Abeta42, soluble and precipitated as amyloid plaques in the brain of old APP/V717I transgenic mice. We tested the hypothesis that aging acted on presenilin1 (PS1) to affect gamma-secretase-mediated production of amyloid peptides by comparing aged APP/V717I transgenic mice to double transgenic mice coexpressing human PS1 and APP/V717I. In double transgenic mice with mutant (A246E) but not wild-type human PS1, brain amyloid peptide levels increased and resulted in amyloid plaques when the mice were only 6-9 months old, much earlier than in APP/V717I transgenic mice (12-15 months old). Mutant PS1 increased mainly brain Abeta42 levels, whereas in aged APP/V717I transgenic mice, both Abeta42 and Abeta40 increased. This resulted in a dramatic difference in the Abeta42/Abeta40 ratio of precipitated or plaque-associated amyloid peptides, i.e., 3.11+/-0.22 in double APP/V717I x PS1/A246E transgenic mice compared with 0.43 +/- 0.07 in aged APP/V717I transgenic mice, and demonstrated a clear difference between the effect of aging and the effect of the insertion of a mutant PS1 transgene. In conclusion, we demonstrate that aging did not favor amyloidogenic over nonamyloidogenic processing of APP, nor did it exert a mutant PS1-like effect on gamma-secretase. Therefore, the data are interpreted to suggest that parenchymal and vascular accumulation of amyloid in aging brain resulted from failure to clear the amyloid peptides rather than from increased production.

Aging↗
PubMedDate not supplied

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.

Animals↗
PubMed2005

CMV-hFasL transgenic mice prevent from experimental autoimmune thyroiditis.

BACKGROUND: Previous studies showed that the role of Fas ligand (FasL) is not consistent in the pathogenesis of autoimmune thyroiditis. This study was designed to investigate the effects of FasL on the pathogenesis of experimental autoimmune thyroiditis (EAT) using CMV-human FasL (hFasL) transgenic mice. METHODS: Transgenic mice ubiquitously expressing hFasL were used as an animal model of EAT by injection of porcine thyroglobulin (pTg). Expression of hFasL was detected by RT-PCR and Western blot. The activity of hFasL transgenic thyrocytes killing Jurket cells was determined. CMV-hFasL transgenic mice and wild type (WT) mice were immunized with pTg and killed 28 days later to evaluate the lymphocytic infiltration of their thyroids. The number of CD4+ and CD8+ lymphocytes from the spleen was detected using FACS. The serum interferon-gamma (IFN-gamma) concentration was measured by ELISA. RESULTS: hFasL expression in the thyroid of CMV-hFasL transgenic mice was confirmed. After co-incubation of Jurket thymocytes with thyroid tissues of CMV-hFasL transgenic mice, the percentage of apoptotic cells in the CMV-hFasL transgenic thyroid group was significantly higher than that of the control WT thyroid group [(23.4 +/- 4.3)% vs (6.6 +/- 2.5)%, P < 0.01]. On day 28 after immunization with pTg, the infiltration index of lymphocytes in thyroids of the CMV-hFasL transgenic mice was significantly lower than that of the WT mice [(1.0 +/- 0.5) vs (2.1 +/- 0.7), P < 0.001]. Moreover, the number of CD4+ and CD8+ lymphocytes of the spleen and serum IFN-gamma concentration were significantly decreased in the CMV-hFasL transgenic mice. CONCLUSIONS: FasL plays an important role in the pathogenesis of autoimmune thyroiditis. Transgenic mice ubiquitously expressing hFasL may strongly inhibit lymphocytic infiltration of the thyroid of EAT and ameliorate the course of this disease.

Animals↗
PubMed1991

Selective up-regulation of macrophage function in granulocyte-macrophage colony-stimulating factor transgenic mice.

Peritoneal and pleural cells from mice transgenic for GM-CSF were studied with regard to their phenotype and functional capacity, and compared with cells from normal littermates. Transgenic mice showed markedly elevated peritoneal and pleural cell counts compared with littermates, and a significantly higher proportion of cells in the transgenic populations were macrophage in phenotype. Transgenic macrophages were larger than the littermate cells, showing abundant foamy cytoplasm and enhanced spreading on plastic. Analysis by flow cytometry showed a more than sixfold increased expression of the macrophage activation markers MAC-2 and MAC-3, but not other markers, on transgenic macrophages. Superoxide production was measured in whole cell populations, both in their basal state and in response to particulate (zymosan) and soluble (PMA) stimuli. Both basal and stimulated superoxide production were markedly elevated in transgenic mice of 12 wk of age, with the largest differences seen in response to PMA. In younger mice, however, only PMA-stimulated superoxide production was significantly greater in transgenic macrophages than in littermate cells and levels of superoxide were generally lower than those seen in 12-wk-old mice. These findings suggest that the enhanced functional capacity of transgenic cells is a maturation-dependent event. In contrast to these findings, drug-dependent cytotoxicity assays performed on cells from 12-wk-old mice revealed no significant differences in killing capacity between the two mouse strains. Taken together these data indicate a selective rather than uniform functional up-regulation in transgenic macrophages compared with their littermates, with a time scale suggestive of a maturational rather than activation process. These findings may provide an indication of the functional macrophage phenotype resulting from long term exposure to GM-CSF in vivo, and help to explain the macrophage-associated pathology seen in GM-CSF-transgenic mice.

Animals↗
PubMed1991

E mu N- and E mu L-myc cooperate with E mu pim-1 to generate lymphoid tumors at high frequency in double-transgenic mice.

Transgenic mice that contain the L-myc gene under the control of the immunoglobulin heavy-chain enhancer (E mu) express the transgene preferentially in T cells, develop thymic hyperplasia and are predisposed to T-cell lymphomas. An analogous E mu N-myc transgene is expressed preferentially in pre-B and B cells and provokes the development of B-cell neoplasias. Animals with an E mu pim-1 construct express the transgene in both B and T cells, but succumb to T-cell lymphomas. Complementation of the E mu N- and L-myc transgenic mice by breeding with E mu pim-1 animals leads to much more rapid development and a dramatically higher incidence of lymphoid malignancies, but the lineage specificity prescribed by the E mu N- and L-myc transgenes is maintained. The different oncogenic potential of myc genes is illustrated by the average latency period of tumor manifestation in double transgenics. Whereas c-myc/pim-1 animals develop pre-B-cell leukemia prenatally, the mean latency period for N-myc/pim-1 and L-myc/pim-1 mice is 36 and 94 days respectively. The N- and L-myc transgenes are expressed at high levels in tumors from double transgenic mice, but expression of the endogenous c- and N-myc genes is undetectable, directly implicating the myc transgenes in the tumor formation process.

Animals↗
PubMed2006

[The establishment of conditional MT transgenic mice model].

OBJECTIVE: To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo. METHODS: Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR. RESULTS: The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding. CONCLUSION: Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.

Animals↗
PubMed1991

Chemically induced tumors in transgenic mice carrying prototype human c-Ha-ras genes.

Three independent transgenic mouse lines carrying human prototype c-Ha-ras genes were established. Approximately 50% of the transgenic offspring of these lines developed spontaneous tumors within 18 months. Types of tumors were restricted to angiosarcomas, skin papillomas and lung or Harderian adenocarcinomas. Interestingly, all angiosarcomas (16/16) had point mutations at the 61st codon of the transgenes. Furthermore, they were also very susceptible to the chemical carcinogens, N-methyl-N'-nitrosourea (MNU) and dimethyl-benzanthracene (DMBA). Within 12 weeks after administration of MNU, the transgenic mice developed forestomach papillomas and then carcinomas very frequently, at the rate of almost 100% and almost all of the tumors had point mutations in their transgenes at the 12th codon from GGC (Gly) to GAC (Asp). All the carcinomas of the forestomach, lung and spleen induced by DMBA had point mutations at the 61st codon from CAG (Gln) to CTG (Leu). Because no somatic point mutations in the transgenes have ever been detected in normal tissues of the affected mice, these mutations seemingly activated the human prototype c-Ha-ras transgene. From these results, it is suggested that the somatic mutation of the human c-Ha-ras transgene plays a causative role in the occurrence of natural tumors and those induced by MNU or DMBA administration in transgenic mice. This transgenic mouse provides a unique screening system for chemicals that induce or suppress the tumorigenesis.

Animals↗
PubMed1990

Expression of a T cell receptor gamma-chain (V gamma 1.1J gamma 4C gamma 4) transgene in mice influences T cell receptor ontogeny and thymic architecture during development.

In this report, we have characterized T cell ontogeny in mice transgenic for the V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) TCR chain gene by analyzing the T cell surface phenotype and microarchitecture of the transgenic lymphoid organs during development. The first wave of V gamma 3J gamma 1C gamma 1 TCR+ thymocytes that appear in the thymus at day 14 of gestation were virtually absent in the TCR gamma 4 transgenic mice. However, comparable levels of total gamma delta+ thymocytes were present in the TCR gamma 4 transgenic mice, suggesting that these thymocytes expressed a transgene receptor. In addition, the appearance of significant numbers of TCR alpha beta+ T cells occurred earlier (day 17 of fetal development) in the transgenic thymus. After birth, thymuses from TCR gamma 4 transgenic mice were characterized by a consistent increase in the number of TCR gamma delta+ thymocytes and a transient increase (between 2 and 4 wk of age) in the absolute number of TCR+ thymocytes (2- to 4-fold) compared with thymuses from normal mice. Immunohistologic analysis of the thymus, spleen, and lymph nodes from 2-day and 3-wk-old transgenic mice showed significant differences to controls only in regions harboring mature, functional T cell subsets which may be the result of bidirectional signaling between lymphocyte subsets (influenced by transgene expression) and stromal elements. The results demonstrate that striking differences, leading to the earlier appearance of mature T cells, occurred in both the fetal and neonatal thymus and periphery of mice that expressed a TCR gamma 4 transgene. These findings are consistent with the hypothesis that expression of the TCR gamma 4 chain gene, early in ontogeny, has a positive influence on the development of the T cell compartment.

Animals↗
PubMed1989

Expression of the rat L-type pyruvate kinase gene from its dual erythroid- and liver-specific promoter in transgenic mice.

The gene for the L-type pyruvate kinase possesses two promoters which are located 500 base pairs apart. The L promoter is specific to liver and regulated by hormones and diet; the L' promoter is specific to erythroid cells. We produced two series of transgenic mice carrying either the entire rat L-pyruvate kinase gene or a minigene devoid of exons two to nine, with 2.7 kilobases of flanking sequences 5' to the cap site of the L' promoter and 1.4 kilobases 3' to the downstream polyadenylation site. In both series the patterns of expression from the two promoters were similar to those of the endogenous rat gene. The rat L promoter was expressed strongly in liver and weakly in kidney and gut of adult transgenic mice. Moreover, it was regulated like the endogenous rat L-pyruvate kinase gene upon hormonal and nutritional adaptation: the level of L-pyruvate kinase mRNA was decreased dramatically by 24 h of starvation, while refeeding a carbohydrate-rich diet strongly stimulated expression of the transgenes. This stimulation was prevented by glucagon. Use of alternative polyadenylation sites in the last exon of the rat L-type pyruvate kinase gene was similar for both types of transgenes and similar to that in rat and not control mice, suggesting that the transgenes contain the sequences that control the choice of polyadenylation site. Transcription of the minigene was higher than that of the entire transgene, probably due to the high copy number of the minigene. At the protein level, rat L subunits encoded by the entire transgene were more abundant than mouse subunits in the liver of adult transgenic mice. In contrast, expression of the rat L' promoter in fetal liver was only 5% of that in fetal rat liver, and we were unable to detect rat L' subunits of pyruvate kinase enzyme in the red blood cells from transgenic mice. Our results suggest that the integrated DNA contains all elements necessary for tissue specificity (L' and L) as well as hormonal and nutritional control (L) of expression of the rat transgene. Nevertheless, a L'-specific activating element may be missing.

Animals↗
PubMed1995

Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus envelope transgenic mice.

Hepatitis B virus (HBV) transgenic mice containing the HBV envelope open reading frame under the transcriptional control of the mouse albumin promoter express hepatitis B surface Ag (HBsAg) in all of their hepatocytes and secrete HBsAg (10 to 40 ng/ml) into the circulation. Because these transgenic mice show no signs of spontaneous liver cell injury or autoimmunity toward the viral (self-) Ag, we asked whether the state of self-tolerance could be reversed by the induction of an acute necroinflammatory liver disease or by immunization with HBV envelope proteins, with the aim of creating a transgenic model for chronic, immune-mediated hepatitis. Our studies indicate that repetitive administration of bacterial LPS, IFN-gamma, or HBsAg-specific CTL, all of which were previously shown to cause liver cell injury and inflammation, does not break tolerance at the T or B cell level, suggesting that the intrahepatic lymphomononuclear cell infiltrate induced by these agents consists of HBsAg-nonspecific cells. The adoptive transfer of HBsAg-primed nontransgenic CD4+ T cells into transgenic mice did not induce anti-HBs autoantibody production by transgenic B cells, even though transgenic B cells were fully responsive to immunization with HBsAg when appropriate T cell help was provided in a nontransgenic environment. Immunization of transgenic mice with purified HBsAg in CFA and repetitive infection with rHBV envelope vaccinia virus led to production of T cell-dependent anti-HBs autoantibodies that cleared HBsAg from the serum, but not to activation of HBsAg-specific CTL. We conclude that HBV envelope transgenic mice are largely tolerant to the transgene product at the T cell but not at the B cell level, and that the activation of an anti-HBs response was not sufficient to induce an autoimmune liver disease in this HBV envelope transgenic mouse model.

Animals↗
PubMed1994

Dependence of increased linear bone growth on age at oMT1a-oGH transgene expression in mice.

We have previously shown that growth hormone (GH) accelerates the rate and extends the duration of linear bone growth in a GH-transgenic mouse model. To determine if this GH effect was temporally regulated, bone lengths and growth plate widths were determined in transgenic mice carrying an ovine metallothionein 1a-ovine growth hormone (oMT1a-oGH) transgene. Transgene expression was initiated in oMT1a-oGH hemizygous transgenic mice by addition of 25 mM ZnSO4 to the water at 21 d of age; littermate control mice were also ZnSO4 supplemented. These mice were maintained on ZnSO4 until 70 d of age at which time the mice were killed and the ulna, humerus, and tibia were collected. Additional transgenic and control mice were stimulated at 21 d and the ZnSO4 stimulus withdrawn after 21 d of treatment and killed at 70 d or stimulated at 28 d with the stimulus withdrawn after 28 d and killed at 70 d. Continuously stimulated transgenic mice had longer bones than those of comparable control mice. The increased bone length in the transgenics was correlated with wider growth plates. Transgene expression initiated at 21 d of age increased bone length to the same or greater extent than that observed for mice with transgene expression initiated at 28 d but maintained for a longer interval thereby indicating that the age of GH initiation is more critical in bone elongation than the duration of GH exposure. The elevated GH effects were independent of circulating thyroid hormones and attainment of puberty. Elevated GH also increased the widths of all growth plate zones to the same extent by increasing cell numbers, rather than enhancing matrix production or individual cellular area.

Aging↗
PubMed1994

Altered lipoprotein metabolism in transgenic mice expressing low levels of a human receptor-binding-defective apolipoprotein E variant.

Transgenic mouse lines were produced that expressed low levels of a receptor-binding-defective variant of human apolipoprotein (apo) E, apoE(Arg112, Cys142). In transgenic mice, the human apoE was produced only by the kidney, whereas endogenous mouse apoE was produced mainly by the liver. The plasma concentration of the transgenic protein was about half that of endogenous apoE. The expression of transgenic apoE did not affect total plasma cholesterol and triglyceride levels, but the distribution of the human variant differed from that of endogenous apoE in the intermediate size and density range, where the transgenic protein accumulated selectively. Immunoblots of agarose gels of lipoprotein fractions showed that the transgenic protein occurred primarily on large alpha-migrating particles (HDL1). This phenomenon was not observed in transgenic mice expressing normal human apoE-3, which distributed like endogenous apoE, suggesting that the defective apoE variant perturbed HDL1 metabolism. In mice fed a high-fat, high-cholesterol diet, the transgenic apoE associated primarily with the apoB-containing lipoproteins. A significantly higher increase in very low density lipoprotein cholesterol was observed in fat-fed transgenics compared to fat-fed nontransgenic mice, suggesting a metabolic perturbation of apoB-containing lipoproteins. Thus, the receptor-binding-defective variant, apoE(Arg112, Cys142), expressed at low levels by the kidney, alters lipoprotein metabolism in transgenic mice, presumably by interfering with apoE-mediated removal of the lipoproteins from circulation.

Animals↗
PubMed1993

Wasting, ischemia, and lymphoid abnormalities in mice expressing T cell-targeted human tumor necrosis factor transgenes.

To evaluate the biologic potential of T cell-specific TNF production in vivo, we have generated transgenic mice constitutively expressing TNF in their T cell compartment. This was achieved by placing a wild-type or a 3'-UTR modified fragment of the human TNF gene under the influence of the T cell-specific, locus control region of the human CD2 gene. Transgenic mice that express human TNF mRNA in T cells develop marked histologic and cellular changes locally in their lymphoid organs and a lethal wasting syndrome associated with widespread vascular thrombosis and tissue necrosis. The extent of pathologic changes and their time of onset appear to reflect levels of transgene expression. Thus, transgenic lines that express the transgene at high levels show both lymphoid organ and systemic abnormalities with wasting. In one transgenic line, mice express lower levels of the transgene and develop normally despite pronounced local lymphoid organ defects, confirming in vivo, the differential potential of localized and systemic TNF action. All pathologic changes could be neutralized by the administration of mAb specific for human TNF. These results demonstrate the important role of T cell-specific TNF production in the development of specific pathology and provide a means by which to evaluate the role of TNF in thymocyte development. Transgenic mice that express TNF constitutively in the T cell compartment offer a unique in vivo system by which to analyze the molecular character of systemic vs contact-dependent and paracrine modes of TNF action. Furthermore, given the species-specific nature of the mouse p75 TNF receptor, it is assumed that the pathology induced by human TNF in these transgenic mice is associated exclusively with p55 TNF receptor signaling. Conceivably, the differential contribution of each of the two TNF receptors in thymus development and TNF-mediated disease can be assessed by comparison of the biologic potential of human vs mouse TNF in the transgenic system developed.

Animals↗
PubMed1993

Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription.

Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.

Animals↗
PubMed1993

Pituitary morphology of transgenic mice expressing bovine growth hormone.

BACKGROUND: In mice transgenic for growth hormone (GH), the ectopic production of foreign GH causes gigantism and strong inhibition of endogenous pituitary GH. In human (h) GH transgenics, morphologic changes occurred not only in somatotrophs but in lactotrophs, corticotrophs, and gonadotrophs as well. To distinguish between changes attributed to somatotrophic effect of hGH from those caused by its lactogenic effects, we studied the pituitary morphology of bovine (b) GH transgenics since bGH has no lactogenic activity. EXPERIMENTAL DESIGN: Pituitaries from transgenic mice and nontransgenic siblings were studied by immunocytochemistry for adenohypophysial hormones, in situ hybridization for GH, prolactin (PRL), and proopiomelanocortin mRNAs, and electron microscopy. RESULTS: Transgenic mice had an increased body weight and a significantly decreased pituitary mass. In both sexes, GH immunoreactive cells were markedly reduced in size and moderately decreased in number, and the GH mRNA signal was lower compared with controls; ultrastructurally, in somatotrophs, the cytoplasmic organelles involved in hormone synthesis were inconspicuous. Males were normoprolactinemic, and lactotrophs showed no morphologic changes. In transgenic females, PRL immunoreactive cells were hypertrophic and appeared to be more numerous. In transgenic males, a mild increase in size and number of follicle-stimulating hormone/luteinizing hormone immunoreactive cells was noted. No changes were evident in corticotrophs and thyrotrophs in either sex. In the intermediate and posterior lobes, many corticotrophs and pituicytes were GH immunoreactive, indicating expression of metallothionein-1 (MT)-bGH transgene. CONCLUSIONS: Pituitaries of MT-bGH transgenic mice contained somatotrophs with morphologic features of inhibition. Endogenous GH production was not completely suppressed as indicated by the presence of GH mRNA and GH immunoreactivity. Females had a mild increase in percentage of PRL immunoreactive cells, and previous biochemical data proved that they were hyperprolactinemic. Substantial differences in pituitary morphology between transgenic MT-bGH mice and MT-hGH animals studied previously can be due to the fact that bGH is purely somatotropic, whereas hGH is both somatotropic and lactogenic.

Adrenocorticotropic Hormone↗