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PubMedDate not supplied

Double recoverable block of function--a molecular control of transgene flow with enhanced reliability.

Despite all the achieved benefits and potential promises from recombinant DNA technology of plants, the potential of transgene spread to wild relatives and to non-transgenic crops is still of a wide-spread concern. We continue to develop recoverable block of function (RBF) technology for gene flow control in transgenic plants. RBF consists of two elements: blocking construct (BC) and recovering construct (RC). Natural expression of the BC (barnase) in embryos and sprouts blocks a physiological function essential for survival or reproduction of the transgenic plant (mRNA synthesis and germination). Artificially induced (heat shock treatment) RC (barstar) recovers the blocked function enabling transgenic plant to reproduce. In natural conditions without artificial induction of RC the transgenic plant can not reproduce itself. However, a single RBF may still fail because of the potential for mutations and gene silencing of the inserted constructs. To minimize the frequency of such an inactivation, we developed a double RBF, in which a single insert comprising two BC, flanking a transgene of interest, was constructed and transferred into tobacco (Nicotiana tabacum (L.)). We used a barstar gene driven by a heat shock or 35S promoter as a RC, and two different promoters were used for barnase genes in the BC. One BC contained a seed germination specific cysteine endopeptidase promoter (BC1) and the other contained the cruciferin promoter (BC2), which is active during fruit development and embryogenesis. Three alternative constructs of double RBF are described, and a segregating two-insert as well as a one-insert cassettes, were compared. One-insert system comprising two BC with different nucleotide sequences but degenerate codons that expressed the same Barnase protein appeared to be the most reliable choice. The biological and molecular data obtained suggest that double RBF is a potent transgene containment technique that can safely be applied in agriculture.

Agriculture↗
PubMed2001

Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice.

BACKGROUND AND AIMS: Chromogranin A (CgA) is a multifunctional acidic protein specifically expressed in neuroendocrine cells. In the stomach, CgA is found predominantly in enterochromaffin-like (ECL) cells, where it is regulated by gastrin. We investigated the ability of a promoter fragment comprising 4.8 kb of 5'-flanking DNA of the mouse CgA (mCgA) gene to direct cell-specific expression as well as gastrin responsiveness in the gastroenteropancreatic neuroendocrine system. METHODS: Two independent lines of mCgA 4.8 kb-luc transgenic mice were created. Transgene expression was assessed by determination of luciferase activity and reverse-transcription polymerase chain reaction analysis of luciferase messenger RNA. Cell specificity of transgene expression was investigated by immunohistochemical analysis. The influence of hypergastrinemia on transgene expression was determined after repeated omeprazole injections. RESULTS: In both transgenic lines, mCgA 4.8 kb-luc expression paralleled the expression pattern of the endogenous CgA gene. ECL cells were identified as the major gastric cell population expressing the transgene. Omeprazole treatment stimulated expression of the transgene and the endogenous CgA gene selectivity in the gastric corpus (3-4-fold). CONCLUSIONS: mCgA 5'-flanking DNA (4.8 kb) contain the major cis-regulatory element(s) required for cell-specific CgA expression in the neuroendocrine system and gastrin-responsiveness in the gastric corpus. Further analysis of the CgA promoter in transgenic studies may elucidate the general molecular mechanisms underlying cell-specific gene expression in the gastroenteropancreatic neuroendocrine system.

Animals↗
PubMed1994

Transgenic animals in male reproduction research.

Although transgenic mouse technology has already been widely used for the study of gene function and regulation in many areas of biomedicine, it has been applied only sporadically to the investigation of testicular function. Nevertheless, the contribution of this experimental approach to the understanding of male reproduction is considerable, not least because of the frequency of infertility in transgenic mice. Transgenic mice can be produced by microinjection of DNA constructs in the male pronucleus of fertilized eggs that are then retransferred into the oviducts of pseudopregnant females and allowed to develop to term. A proportion of the offspring have the foreign DNA sequences permamently integrated into the genome and thus become transgenic. In this way it is possible to obtain either the over-expression of genes, which can be targeted to the testis using testis-specific promoters, or to effect interruption of the functional integrity of genes by insertional mutagenesis. The regulation of gene expression in vivo can be studied by producing transgenic mice where the transgene is composed of the regulatory sequences of a gene of interest driving the expression of a reporter gene. Specific genes can be "knocked out" by homologous recombination. This article reviews the contribution of the transgenic approach to the following areas of male reproduction: the identification of factors involved in sex determination and development of the reproductive tract; the study of the function and expression of genes important for spermatogenesis and male reproduction; the identification of genes involved in spermatogenesis and of genomic sequences directing the expression of a transgene in the testis; the study of the function of specific reproductive tissues or cells in vivo; oncogenesis in reproductive tissues; the creation of cell lines suitable for in vitro studies; gene therapy.

Animals↗
PubMed2005

Epstein-Barr virus nuclear antigen 1 does not induce lymphoma in transgenic FVB mice.

The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was evaluated in three transgenic FVB mouse lineages. EBNA1 was expressed at a higher level in transgenic B220(+) splenocytes than in EBV-infected lymphoblastoid cell lines. EBNA1 was also expressed in B220(-) transgenic splenocytes and thymocytes. Before killing and assessments at 18-26 months, EBNA1-transgenic mice did not differ from control mice in mortality. At 18-26 months EBNA1-transgenic mice did not differ from littermate control in ultimate body weight, in spleen size or weight, in lymph node, kidney, liver, or spleen histology, in splenocyte fractions positive for cluster of differentiation (CD)3epsilon, CD4, CD8, CD62L, B220, CD5, IgM, IgD, MHC class II, CD11b, or CD25, or in serum IgM, IgG, or total Ig levels. Lymphomas were not found in spleens or other organs of 18- to 26-month-old EBNA1-transgenic (n=86) or control (n=45) FVB mice. EBNA1-transgenic lineages had a higher pulmonary adenoma prevalence than did littermate controls (39% versus 7%). However, the adenoma prevalence was not higher in EBNA1-transgenic mice than has been described for FVB mice, and EBNA1 was not expressed in normal pulmonary epithelia or adenomas.

Adenomatosis, Pulmonary↗
PubMed1988

Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM.

Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).

Animals↗
PubMed1989

Isotype switching by a microinjected mu immunoglobulin heavy chain gene in transgenic mice.

Immunization of transgenic mice carrying an immunoglobulin mu heavy chain resulted in a response dominated by expression of the transgene variable region. Unexpectedly, in a large proportion of the antibody produced by immunized mice, the transgene variable region was associated with IgG rather than IgM. This demonstrates that the transgene can undergo an isotype switch. Four transgenic founder lines all exhibited transgene isotype switching despite the likelihood of random chromosomal integration of the transgene. In addition one of the lines was analyzed by breeding studies and the transgene was found to be genetically unlinked to the immunoglobulin heavy chain (Igh) locus. These results indicate that a precise chromosomal location is not required for isotype switching and suggest the possibility that the isotype switching process can occur interchromosomally.

Animals↗
PubMed1991

Transgenic mice susceptible to poliovirus.

Poliovirus-sensitive transgenic mice were produced by introducing the human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic mice just as in tissues of humans. The transgenic mice are susceptible to all three poliovirus serotypes, and the mice inoculated with poliovirus show clinical symptoms similar to those observed in humans and monkeys. Rabbit antipoliovirus serum detects the antigens mainly in motor neurons in the anterior horn of the spinal cord and in nerve cells in the medulla oblongata and pons of the paralyzed transgenic mice. Therefore, cell types sensitive to poliovirus in the central nervous system of the transgenic mice appear to be identical to those of humans and monkeys. Furthermore, many more doses of oral poliovirus vaccine strains than of the virulent strains are required to cause paralysis in the transgenic mice. This may reflect the observation that the virulent strain multiplies more efficiently in the central nervous system than the attenuated strain. Thus, the transgenic mice may become an excellent new animal model to study molecular mechanisms of pathogenesis of poliovirus and to assess oral poliovirus vaccines.

Animals↗
PubMed1993

Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene.

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.

Amino Acid Sequence↗
PubMed1993

Comparison of somatic mutation in a transgenic versus host locus.

Somatic mutations can now be quantified in almost any cell type in mice carrying bacterial genes in a lambda phage shuttle vector. Mutations induced in vivo are detectable ex vivo, after packaging host-cell DNA into phage that are grown on suitable bacteria. However, the transgenic DNA differs from many host loci in several ways: it (i) is prokaryotic DNA, (ii) is present in multiple tandem copies, and (iii) is heavily methylated and probably not expressed. Thus, mutation of a transgene may not be a suitable model of the host loci, which are eukaryotic, unique, and expressed. To test the relevance of the transgene mutation model, the frequencies of the bacterial lacI+ to lacI- mutations induced in half of the small intestine were compared with the frequencies of the host Dlb-1b to Dlb-1a mutations induced in the other half. The loci responded similarly to ethyl nitrosourea (ENU) with respect to the animal's age and sex, sex of the parent transmitting the transgene, and expression time. ENU dose-response curves were similar. Furthermore, no difference was found at the Dlb-1 locus between transgenic and nontransgenic siblings. In contrast, x-rays induced few lacI mutations but many Dlb-1 mutations. Probably few large deletions are detectable at lacI, but many are detectable at Dlb-1. If so, an important class of mutation is not readily detected in these transgenic mice. With this exception, the transgene and host gene responded similarly in this somewhat limited trial, as is necessary if the transgenic mice are to be a useful model.

Animals↗
PubMed1994

Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region beta-chain T-cell repertoire and mediates an HLA-DR-restricted immune response.

Inherited susceptibility to rheumatoid arthritis is associated with genes encoding the human major histocompatibility complex class II molecule HLA-DR4. To study the immune function of HLA-DR4 and attempt to generate a murine model of rheumatoid arthritis we have produced triple transgenic mice expressing HLA-DRA*0101, -DRB1*0401, and human CD4. The expression of the HLA transgenes is driven by the promoter of the murine major histocompatibility complex class II I-E alpha gene and was found on murine cells that normally display major histocompatibility complex class II molecules. The expression of the human CD4 transgene is driven by the murine CD3 delta-promoter, and therefore its gene product was found on cells that express murine CD3. In contrast to other HLA-DR and HLA-DQ transgenic mouse lines, the transgenes are functional in our mice. In H-2 I-E-negative transgenic mice, T cells expressing variable region beta chain (V beta) 3, 5, 6, 7, 9, 11, 12, or 13 were either absent or significantly reduced, in contrast to H-2 I-E-negative nontransgenic littermates. In addition, the peptide antigen influenza A virus hemagglutinin 307-319, which binds to the HLA-DRA*0101/-DRB1*0401 heterodimer with high affinity and induces an HLA-DR-restricted and CD4+ T-cell response in humans, also induced a T-cell response in the triple transgenic mice but not in nontransgenic littermates. Thus, these transgenic mice should permit extensive testing of the antigen-presentation capabilities of the HLA-DRA*0101/-DRB1*0401 molecule.

Animals↗
PubMed1994

Epidermal expression of intercellular adhesion molecule 1 is not a primary inducer of cutaneous inflammation in transgenic mice.

Keratinocytes at sites of cutaneous inflammation have increased expression of intercellular adhesion molecule 1 (ICAM-1), a cytokine-inducible adhesion molecule which binds the leukocyte integrins LFA-1 and Mac-1. Transgenic mice were prepared in which the expression of mouse ICAM-1 was targeted to basal keratinocytes by using the human K14 keratin promoter. The level of constitutive expression attained in the transgenic mice exceeded the peak level of ICAM-1 expression induced on nontransgenic mouse keratinocytes in vitro by optimal combinations of interferon gamma and tumor necrosis factor alpha or in vivo by proinflammatory stimuli such as phorbol 12-myristate 13-acetate. In vitro adhesion assays demonstrated that cultured transgenic keratinocytes were superior to normal keratinocytes as a substrate for the LFA-1-dependent binding of mouse T cells, confirming that the transgene-encoded ICAM-1 was expressed in a functional form. However, the high level of constitutive ICAM-1 expression achieved on keratinocytes in vivo in these transgenic mice did not result in additional recruitment of CD45+ leukocytes into transgenic epidermis, nor did it elicit dermal inflammation. Keratinocyte ICAM-1 expression also did not potentiate contact-hypersensitivity reactions to epicutaneous application of haptens. The absence of a spontaneous phenotype in these transgenic mice was not the result of increased levels of soluble ICAM-1, since serum levels of soluble ICAM-1 were equal in transgenic mice and controls. We conclude that elevated ICAM-1 expression on keratinocytes cannot act independently to influence leukocyte trafficking and elicit cutaneous inflammation.

Animals↗
PubMed1995

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

Animals↗
PubMed1996

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

Animals↗
PubMed1997

Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy.

Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its own cis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.

Animals↗
PubMed1995

Bovine oxytocin transgenes in mice. Hypothalamic expression, physiological regulation, and interactions with the vasopressin gene.

To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.

Animals↗
PubMed1996

Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.

The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/- 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism.

Animals↗
PubMed1997

Phosphoenolpyruvate carboxykinase (GTP) gene transcription and hyperglycemia are regulated by glucocorticoids in genetically obese db/db transgenic mice.

The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and the cis-acting insulin response sequence (IRS, -416/-407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express -460 or -2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at the db locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 +/- 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, -2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabetic db/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that -460 bp of 5'-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obese db/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in the db/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (-416/-407) promoter binding site.

Animals↗
PubMed2001

Regulation and activity of the human ABCA1 gene in transgenic mice.

The ABCA1 transporter is one of the limiting steps in cellular cholesterol efflux. To study the expression and activity of the human ABCA1 gene in vivo we have examined mice containing two human BAC transgenes with different 5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including 70 kb upstream of the previously defined exon 1, demonstrated a pattern of tissue-specific expression mimicking that of the endogenous mouse gene. Compared with macrophages from control mice, macrophages from these transgenics had increases in apoA-I cholesterol efflux heightened in response to increases in cell cholesterol content. The observed increase in macrophage apoA-I-mediated cholesterol efflux was not accompanied by alterations in plasma high density lipoprotein in the transgenics. Although mice containing a smaller 171-kb human BAC transgene, lacking the previously described exon 1 and ABCA1 promoter, did not express human ABCA1 in macrophages, they did express the human transgene in liver at levels comparable with those of the orthologous mouse gene. Analysis by 5' rapid amplification of cDNA ends of liver mRNA from these animals revealed a new ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis of human tissue revealed that exon 1A containing transcripts accounted for a high proportion of the ABCA1 mRNAs present in human liver. This analysis of ABCA1 transgenics showed that the expression of human ABCA1 transgenes can result in increased cholesterol efflux from macrophages, unaccompanied by changes in plasma high density lipoprotein, and identified a new ABCA1 promoter in humans.

ATP Binding Cassette Transporter 1↗