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Transgenic mice models in autoimmunity--discussion.

Transgenic mice are an important in vivo model for studying the function of single genes. Specific induction and tissue specific expression of the inserted genes are the great advantages of this system. However, there a risks in constructing transgenic mice and the interpretation of the experimental data. To prevent artefacts and to optimize the transgenic model, the experimental systems have to fulfill the following presets: 1. Experiments has to be done with a stable transgenic line and not with the first heterogeneous transgeniced generation, because they differ in their site of gene insertion and in the cellular response. 2. The number of experiments have to be statistically sufficient. Often only a few number of experiments are done, due to the bad reproduction of transgenic mice. 3. Health of transgenic mice has to be proven, since the transgenic animals are under laboratory conditions and not under professional breeding conditions. 4. As a control there should be a transgenic mouse with a comparable pseudogene insertion beside the normal littermate control, to exclude artefacts by the simple gene insertion. If all of these presets are fulfilled, a transgenic mice model will be one of the best experimental systems.

Animals↗

The peripheral deletion of autoreactive CD8+ T cells induced by cross-presentation of self-antigens involves signaling through CD95 (Fas, Apo-1).

Recently, we demonstrated that major histocompatibility complex class I-restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8+ T cells. In these studies, naive ovalbumin (OVA)-specific CD8+ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8+ T cells in the whole animal.

Animals↗

Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

Animals↗

Delayed abscission and shorter Internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato.

Stable transformation of tomato (Lycopersicon esculentum cv Ailsa Craig) plants with a construct containing the antisense sequence for the receiver domain and 3'-untranslated portion of the tomato ethylene receptor (LeETR1) under the control of an enhanced cauliflower mosaic virus 35S promoter resulted in some expected and unexpected phenotypes. In addition to reduced LeETR1 transcript levels, the two most consistently observed phenotypes in the transgenic lines were delayed abscission and reduced plant size. Fruit coloration and softening were essentially unaffected, and all the seedlings from first generation seed displayed a normal triple response to ethylene. Two independent lines with a single copy of the transgene and reduced LeETR1 transcript accumulation were selected for detailed phenotypic analysis of second generation (R1) plants. Delayed abscission, shorter internode length, and reduced auxin movement all correlated with the presence of the transgene and the degree of reduced LeETR1 transcript accumulation. No significant differences were noted for fruit coloration or fruit softening on R1 plants and all seedlings from R1 and R2 seed displayed a normal triple response. LeETR2 transcript accumulation was only slightly reduced in the R1 plants compared with azygous plants, and LeETR3 (NR) transcript levels appeared to be unaffected by the transgene. We propose that ethylene signal transduction occurs through parallel paths that partially intersect to regulate shared ethylene responses.

Biological Transport↗

Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens T-DNA oncogenes.

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5' end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Agrobacterium tumefaciens↗

Treatment with propionic and butyric acid enhances expression variegation and promoter methylation in plant transgenes.

Two phenotypic marker genes (A1 and GUS) were employed to monitor the influence of small chain fatty acids on transgene expression in petunia and tobacco. In plants homozygous with respect to the A1 transgene, which normally carry red flowers due to A1 expression, fatty acid treatment induced a range of variegated and white pigmentation patterns which persisted for several months after terminating the treatment. The inhibitory effect was clearly less pronounced for heterozygous plants of the same transgenic line. In all cases the reduction of transgene activities correlated with an increase in transgene promoter methylation. Contrary to evidence reported for mammals and Drosophila, we do not observe an increase in gene expression, but an enhancement of DNA methylation and epigenetic variegation. The inhibition of transgene activity was also observed in several tobacco transformants cultured on fatty acid containing media. Some tobacco transformants carrying Gus-coding regions driven by either 35S or 1'2' promoters responded with a significant reduction in GUS activity. This study suggests that, rather than exerting a general response on all chromatin regions, fatty acids appear to affect genes in a labile epigenetic surrounding or all genes in a susceptible chromatin configuration. Thus, application of these agents may be useful to screen and monitor transgenes prone to epigenetic instabilities.

Acetylation↗

Brain tumours and lymphomas in transgenic mice that carry HTLV-I LTR/c-myc and Ig/tax genes.

The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.

Animals↗

scl, a gene frequently activated in human T cell leukaemia, does not induce lymphomas in transgenic mice.

The scl gene is implicated in human T cell acute lymphoblastic leukaemia (T-ALL) through its involvement in the t(1;14)(p32;q11) chromosomal translocation and, more frequently, as a result of a tumour-specific interstitial deletion on chromosome 1. The consequence of both these chromosomal alterations is overexpression of scl in the leukaemic cells. Despite the strong inference of a role in human T-ALL, scl has not yet been demonstrated to be causally involved in neoplastic transformation. We attempted to do this by generating transgenic mice in which scl expression was directed to the T cell lineage using the CD2 enhancer and the strong SR alpha viral promoter (CD2-scl mice). Three transgenic lines, all of which expressed the scl transgene at a high level, were bred and analysed. No alterations in T cell development were seen in the mice. Unexpectedly CD2-scl mice did not develop tumours, nor did the transgene enhance tumourigenesis by Moloney murine leukaemia virus. These findings throw into question the mechanism by which aberrant scl expression contributes to T cell leukaemogenesis.

Animals↗

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development.

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.

Animals↗

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis.

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.

Amino Acid Sequence↗

Induction of the enzyme aldose reductase in a lens epithelial cell line from a transgenic mouse.

A lens epithelial cell line established from a transgenic mouse synthesizes high levels of the enzyme aldose reductase which converts sugars to polyols. This enzyme has been implicated in the formation of sugar cataracts in animals and with diabetic complications in man. The mouse aldose reductase has been characterized and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis has an apparent molecular mass of 38,000, similar to the enzyme in rat and man. The cellular enzyme is inhibited by two aldose reductase inhibitors: Sorbinil (IC50 = 1.8 X 10(-7) M) and Alcon 1576 (IC50 = 7.8 X 10(-8) M). The amount and the specific activity of the aldose reductase can be further increased in the cells by raising the osmolarity of the medium to 500 mOSM. Although the amount of aldose reductase is increased approximately sevenfold under these conditions, alpha-crystallin, one of the main lens specific proteins, remained at about the same concentration. No detectable increase in sorbitol was found within the cells, in contrast to published reports on renal cells in which this polyol increases under similar hyperosmotic conditions; however, in the lens cells there was a five-fold increase in the inositol content, suggesting that this polyol rather than sorbitol may be used to compensate for some of the changes in the osmolarity. The induction of the enzyme aldose reductase without the apparent accumulation of its product suggests a complex mechanism for osmoregulation in the lens cells.

Aldehyde Reductase↗

Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis.

Aminoalcoholphosphotransferases (AAPT, EC 2.7.8.1 and EC 2.7.8.2) catalyze the transfer of CDP-aminoalcohols to sn-1, 2 diacylglycerol (DAG) to form phosphatidylaminoalcohols with the release of CMP. The Brassica napus L. AAPT1 gene (designated BnAAPT1) was identified from cDNA libraries of seedlings and developing seeds. Functional characterization was accomplished by heterologous expression of BnAAPT1 in a yeast strain deficient in AAPT activities. BnAAPT1 exhibited a greater preference for utilizing CDP-choline as a substrate with Vmax of 35 [14C]phosphatidylcholine nmol h(-1) mg(-1) protein and apparent Km of 32 microM while CDP-ethanolamine had a Vmax of 13 [14C]phosphatidylethanolamine nmol h(-1) mg(-1)protein and an apparent Km of 127 microM. The enzyme was activated by Mg2+, Mn2+ and phospholipid mixtures, and inhibited by Ca2+. A CDP-alcohol phosphotransferase motif, Asp99-Gly100-(X2)-Ala103-Arg104-(X8)-Gly113-(X3)-Asp117-(X3)-Asp121, was completely conserved in BnAAPT1 and its catalytic role was confirmed by scanning alanine mutagenesis. Over-expression of BnAAPT1 under the control of the double 35S promoter in transgenic Arabidopsis thaliana (L.) Heynh. plants led to elevated levels of the corresponding transcript and enzyme activity. In four of the high over-expression transgenic lines, phospholipid and fatty acid composition analyses revealed that chloroplastidic and extrachloroplastidic membranes isolated from transgenic leaves had about a 25% increase in phosphatidylcholine and in the proportions of polyunsaturated fatty acids [18:2+18:3], relative to the control. There were also consistent, but small differences observed in the proportions of 18:3 in transgenic green siliques and in 20:1 in mature transgenic seeds of these lines. Induction of Arabidopsis AAPT transcription in response to (+)-abscisic acid and low-temperature treatments, and the cold tolerance in BnAAPT1 transgenic seedlings implies that AAPT may play a role in resistance to damage at low growth temperatures.

Abscisic Acid↗

Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development.

Transcriptional regulation of Arabidopsis thaliana (L.) Heynh. phytochelatin synthase (AtPCS1) by cadmium (Cd) was analyzed at various stages of plant development using transgenic Arabidopsis and wild-type plants. Histochemical analysis of beta-glucuronidase (GUS) activity in transgenic lines carrying a uidA gene driven by a 2.0-kb AtPCS1 promoter revealed higher GUS activities in 5-day-old seedlings subjected to 50 microM Cd treatment for 5 days, beginning at seed germination, than in non-treated plants. This high level of GUS activity gradually decreased as plants continued their growth until no differences were observed between Cd-treated and non-treated transgenic plants. The observed GUS activity due to Cd treatment during the early stage of plant development corresponded with induction of AtPCS1 mRNA as confirmed by RNA blot analysis of wild-type Arabidopsis. The steady-state level of AtPCS1 mRNA increased by 2-fold in 5-day-old Cd-treated wild-type Arabidopsis compared to non-treated seedlings. Moreover, AtPCS1 protein levels increased following Cd treatment as observed in western blot analysis of transgenic Arabidopsis lines carrying a C-terminal FLAG-tagged AtPCS1 genomic DNA driven by a 2.0-kb AtPCS1 promoter. The transcriptional regulation of AtPCS1 by Cd during the early stage of seedling development seemed to be correlated with a higher Cd sensitivity in cad2, an Arabidopsis mutant deficient in phytochelatin synthesis, during early stages of plant development. This was supported by our finding that Cd sensitivity in cad2 was reduced as plants continued their growth, and was comparable to that of wild-type plants.

Aminoacyltransferases↗

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana.

Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana.

Arabidopsis↗

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield.

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T(5)) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6-21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.

Arabidopsis↗

Evidence for cyclophosphamide-induced gene conversion and mutation in mouse germ cells.

Cyclophosphamide (CP) is a widely used antineoplastic drug. It tests positive in several genotoxicity assays, including those with endpoints such as chromosomal aberrations in mammalian cells, mitotic recombination in Drosophila melanogaster, and dominant lethal mutations in rodents. We have explored the effects of CP on genome stability of mouse (Mus domesticus) spermatogenic cells, using a recombination-based transgenic assay called MUSCATEER. In this system, intrachromosomal gene conversion events between two mutually defective lacZ genes generates beta-galactosidase activity in spermatids. The frequency of gene conversion events is determined by scoring spermatids stained with the lacZ substrate, X-gal. A dose-dependent induction of lacZ-positive spermatids was observed following single intraperitoneal CP exposures of 10, 100, and 200 mg/kg. At 200 mg/kg, there was a 25-fold increase over baseline. Treatment of a control transgenic line containing only a frame-shifted lacZ transgene provided an indication that CP also induced reversion mutations. The timing of the response indicated that the induction of recombination and/or mutation occurred primarily in meiotic stage cells. These results demonstrate potent germline mutagenicity of CP, and validate the utility and sensitivity of genetic recombination as a rapid indicator of genotoxicity in whole animals.

Animals↗

The Arabidopsis transposon Tag1 is active in rice, undergoing germinal transposition and restricted, late somatic excision.

Tag1 is an autonomous transposable element of Arabidopsis thaliana that displays tight developmental control of its excision during shoot development. To determine how Tag1 behaves in a monocotyledonous species, Tag1 was inserted in a 35S-GUS marker gene and the construct was introduced into rice. Tag1 showed somatic excision activity in four out of eleven transgenic lines examined. In leaves, excision was primarily restricted to vascular bundles and produced sectors composed of only a few cells. Excision events in flowers occurred predominantly in or near the major veins of the palea and lemma to produce small sectors. In roots, small sectors were evident, but they were few in number. These data show that the timing of Tag1 excision during rice shoot development is late and mimics the late excision behavior of Tag1 in Arabidopsis. One of the transgenic rice lines, which had a high frequency of somatic excision, produced several germinal revertants, one of which was characterized by a new Tag1 insertion band. The pattern of Tag1 transcripts and the footprint sequences left behind after excision in rice were found to be very similar to those in Arabidopsis. These results show that key properties of Tag1 transposition and behavior are conserved between monocots and dicots and that Tag1 has the potential to serve as an insertional mutagen in rice.

Arabidopsis↗

Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is a pleiotropic protein which has been characterized extensively both biochemically and functionally. It is the only one of the identified latent protein-encoding genes to be consistently expressed in viral-associated endemic Burkitt's lymphoma cells. As such, it is the only candidate viral protein to possibly perform a maintenance function in the tumour pathology. Despite this, no oncogenic activity has been attributed to the protein in tissue culture assays. The experiments described here were initiated to explore the activity of the protein in B cells in vivo. EBNA-1 transgenic mice were generated with transgene expression directed to the B cell compartment using the mouse Ig heavy chain intron enhancer. Transgene expression was demonstrated in the lymphoid tissues of mice of two independent lines. Transgenic positive mice of both lines succumb to B cell lymphoma. The B cell tumours are monoclonal, frequently of follicular centre cell origin and remarkably similar to those induced by transgenic c-myc expression. These results demonstrate that EBNA-1 is oncogenic in vivo and suggest that the gene product may play a direct role in the pathogenesis of Burkitt's lymphoma and possibly other EBV-associated malignancies.

Animals↗