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PubMed1995

Commitment and maintenance of the alpha beta and gamma delta T cell lineages.

We have generated transgenic mice using a rearranged, functional TCR-gamma gene that lacks the C gamma 1 3' silencer element, together with a TCR-delta cDNA construct designed for expression in lymphoid cells. All transgenic mice that descended from nine founders expressed alpha beta T cells and elevated levels of gamma delta T cells at various ratios. This observation does not support the proposal that alpha beta T cells are generated from T precursors in which C gamma 1 genes have been repressed via the cis-acting 3' silencer element, but it supports the idea that lineage commitment occurs independently of TCR gene expression. In seven transgenic lines, despite the absence of the 3' C gamma 1 silencer on the gamma transgene, the transgenic TCR is expressed only in gamma delta and not in alpha beta T cells. In the other two lines, in addition to T cells that express either the alpha beta or gamma delta TCR, a substantial fraction of T cells expresses both alpha beta and gamma delta TCR. These two lines also carry the highest number of copies of TCR-gamma transgene. However, in general, there is no correlation between the copy number of the gamma transgene and the fraction of T cells expressing the gamma delta TCR. The implications of these data are discussed.

Aging↗

PubMed2003

The Arabidopsis thaliana rlp mutations revert the ectopic leaf blade formation conferred by activation tagging of the LEP gene.

Activation tagging of the gene LEAFY PETIOLE (LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole (rlp lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups. The class III rlp lines also display altered leaf development in a wild-type (non-transgenic) background, and are probably mutated in genes that affect shoot or leaf development. The rlp lines of classes I and II, which represent the majority of revertants, do not affect leaf blade outgrowth in a wild-type (non-transgenic) background. This indicates that LEP regulates a subset of the genes involved in the process of leaf blade outgrowth, and that genetic and/or functional redundancy in this process compensates for the loss of RLP function during the formation of the wild-type leaf blade. More detailed genetic and morphological analyses were performed on a selection of the rlp lines. Of these, the dominant rlp lines display complete reversion of (1) the leafy petiole phenotype, (2) the reduction in the number of rosette leaves and (3) the slower leaf initiation rate caused by the activation-tagged LEP gene. Therefore, these lines are potentially mutated in genes for interacting partners of LEP or in downstream regulatory genes. In contrast, the recessive rlp lines exhibit a specific reversion of the leafy petiole phenotype. Thus, these lines are most probably mutated in genes specific for the outgrowth of the leaf blade. Further functional analysis of the rlp mutations will contribute to the dissection of the complex pathways underlying leaf blade outgrowth.

Arabidopsis↗

PubMed2005

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction.

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.

Animals↗

PubMed2003

A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice.

Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.

Animals↗

PubMed1997

Tau phosphorylation in transgenic mice expressing glycogen synthase kinase-3beta transgenes.

In order to investigate the effect on tau of manipulating glycogen synthase kinase (GSK)-3beta activity in the brain, we created transgenic mice harbouring wild-type GSK-3beta genes or a mutant GSK-3beta that is predicted to be more active. Transgene-derived mRNAs were detected in the brains of a number of the transgenic mouse lines and several of these transgenic lines displayed transgenic GSK-3beta activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3beta activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3beta is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3beta were obtained and it is possible that high levels of GSK-3beta activity are lethal.

Animals↗

PubMed1995

Somatic intrachromosomal homologous recombination events in populations of plant siblings.

Intrachromosomal homologous recombination in whole tobacco plants was analyzed using beta-glucuronidase as non-selectable marker. We found that recombination frequencies were additive for transgenes in allelic positions and could be enhanced by treatment of plants with DNA-damaging agents. We compared the patterns of distribution of recombination events of different transgenic lines of tobacco and Arabidopsis with the respective Poisson distributions. Some lines showed Poisson-like distributions, indicating that recombination at the transgene locus was occurring in a random fashion in the plant population. In other cases, however, the distributions deviated significantly from Poisson distributions indicating that for specific transgene loci and/or configurations recombination events are not randomly distributed in the population. This was due to overrepresentation of plants with especially many as well as especially few recombination events. Analysis of one tobacco line indicated furthermore that the distribution of recombination events could be influenced by treating the seedlings with external factors. Our results suggest that different plant individuals, or parts of them, might exhibit different transient 'states' of recombination competence. A possible model relating 'recombination silencing' and transcription silencing to heterochromatization of the transgene locus is discussed.

Arabidopsis↗

PubMed2002

Down-regulation of caffeic acid o-methyltransferase in maize revisited using a transgenic approach.

Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven beta-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%-30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brown-midrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.

Coumaric Acids↗

PubMed2003

Identification of a novel DNase I hypersensitive site within the far upstream region of the human HLA-DRA gene.

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.

5' Flanking Region↗

PubMed1993

Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron.

Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

Animals↗

PubMed1999

Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain.

Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, differential glutamatergic responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as 3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal "stubs," and Abeta(40) and Abeta(42) peptides in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected, the Abeta(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late neuropathological and clinical aspects of Alzheimer's disease.

Alzheimer Disease↗

PubMed1990

Cyclic hair-loss and regrowth in transgenic mice overexpressing an intermediate filament gene.

We have produced transgenic mice containing up to 250 copies of a sheep wool intermediate filament keratin gene to study the effect of its expression on hair structure and development. Several transgenic lines expressed the gene and in the one containing 250 transgenes, a pattern of hair-loss and regrowth was stably established. Successive waves of hair growth follow periods of denuding like the natural progression of hairs in the mouse hair cycle. By in situ hybridization we have shown that the sheep transgenes are expressed at the correct stage in mouse hair development and at a high level. The transgenic hairs contain not only an elevated level of intermediate filament keratin protein but also a decreased level of the filament-associated proteins. This imbalance disrupts the normal ordered array of these proteins in the cells of the hair cortex and leads to weakened fibres which are prematurely lost.

Amino Acids↗

PubMed2002

Wound-induced gene expression of putrescine N-methyltransferase in leaves of Nicotiana tabacum.

Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the biosynthesis of pyrrolinium ring-containing alkaloids. Earlier studies have indicated that PMT gene expression is restricted to root tissue in Solanaceus plant species. During the analysis to further elucidate factors that govern the regulation of alkaloid synthesis, evidence was found for a novel expression pattern dictated by the 5'-flanking region of at least two members of the PMT-gene family. A 627-bp DNA fragment upstream of the NtPMT3 gene was fused to the beta-glucuronidase (GUS) reporter gene and used to produce stable transgenic lines of Nicotiana tabacum. Fluorometric and histochemical assays conducted on transgenic plants indicated high expression levels in root tissue and, in agreement with previous studies, no expression was detected in leaves. However, expression was observed in leaves when they were mechanically wounded. This expression was highly localized around the wound site and showed little evidence of long distance signaling, including lack of responsiveness to jasmonic acid. Expression was transient, with maximum levels immediately after wounding and diminishing after approximately 2-4 h. RT-PCR analysis of mRNA isolated from wild-type plants also indicated upregulation of PMT expression in leaves upon wounding as well as very low transcript levels in unwounded leaves. Low levels of PMT activity were detected in leaf tissue, which did not increase significantly upon wounding.

Gene Expression Regulation, Enzymologic↗

PubMed2000

The low molecular mass PsbW protein is involved in the stabilization of the dimeric photosystem II complex in Arabidopsis thaliana.

Arabidopsis thaliana plants have been transformed with an antisense gene to the psbW of photosystem II (PSII). Eight transgenic lines containing low levels of psbW mRNA have been obtained. Transgenic seedlings with low contents of PsbW protein (more than 96% reduced) were selected by Western blotting and used for photosynthetic functional studies. There were no distinct differences in phenotype between the antisense and wild type plants during vegetative period under normal growth light intensities. However, a sucrose gradient separation of briefly solubilized thylakoid membranes revealed that no dimeric PSII supracomplex could be detected in the transgenic plants lacking the PsbW protein. Furthermore, analysis of isolated thylakoids demonstrated that the oxygen-evolving rate in antisense plants decreased by 50% compared with the wild type. This was found to be due to up to 40% of D1 and D2 reaction center proteins of PSII disappearing in the transgenic plants. The absence of the PsbW protein also altered the contents of other PSII proteins to differing extents. These results show that in the absence of the PsbW protein, the stability of the dimeric PSII is diminished and consequently the total number of PSII complexes is greatly reduced. Thus the nuclear encoded PsbW protein may play a crucial role in the biogenesis and regulation of the photosynthetic apparatus.

Amino Acid Sequence↗

PubMed1993

Brain and liver targeted overexpression of O6-methylguanine DNA methyltransferase in transgenic mice.

O6-Methylguanine DNA methyltransferase (MGMT; EC 2.1.1.63) is an unusual DNA repair protein in that it directly and specifically repairs a premutagenic DNA lesion without involving other proteins. MGMT removes the alkyl group from O6-alkylguanine in DNA in a unique stoichiometric reaction by accepting the alkyl group on a cysteine residue. The intracellular level of MGMT varies among tissues and appears to be inversely correlated to tissue-specific tumorigenesis induced by monofunctional alkylating agents. Because MGMT acts in solo, genetic manipulation of its expression may provide valuable insight into its contribution to cellular resistance to alkylation toxicity and to tumor induction. The human MGMT full length cDNA has been fused with a portion of the human transferrin (TF) 5'-flanking region (TF/MGMT). Transgenic founder mice were produced carrying the TF/MGMT transgene and then bred to establish stable transgenic lines. Human MGMT transcripts were specifically expressed in abundance in transgenic brain and liver tissues. In vitro MGMT assays revealed approximately 150-fold and approximately 25-fold increases in MGMT activity in transgenic brain and liver extracts respectively. Western blot analysis confirmed that human MGMT protein is specifically synthesized in transgenic brain and liver tissues.

Animals↗

PubMed1993

Threshold detection of self-antigen/MHC class II complexes formed in vivo: constitutive presentation of an immunodominant epitope of hen egg lysozyme (HEL) in HEL-transgenic mice.

Hen egg lysozyme (HEL)-specific T hybridomas were used to study the presentation of HEL as a self component in tolerant, HEL-transgenic mice expressing different serum levels of HEL (AL3, 3-7 nM: ML5, 1-3 nM). Constitutive presentation of the HEL46-61 determinant was detectable in antigen presenting cell (APC)-enriched preparations of spleen, thymus, and lymph node from AL3 transgenic mice. The most likely cell responsible for constitutive presentation of HEL antigen was a non-B cell. In addition, constitutive presentation of HEL46-61 was clearly evident in thymic rosettes (complexes of macrophages or dendritic cells and lymphoid cells) derived from AL3 HEL-transgenic mice, but not from ML5 HEL-transgenic mice. In contrast to the HEL46-61 determinant, constitutive presentation of the HEL25-43 and HEL112-129 determinants was not detectable on APC-enriched preparations from either of the HEL-transgenic lines tested. Thus, although T cell tolerance was evident in transgenic mice expressing a range of serum HEL concentrations, constitutive presentation of processed HEL antigen was not detectable on APCs from mice expressing less than 3-7 nM. This threshold of detectable antigen presentation is consistent with the notion that the concentration of nominal antigen needed for tolerizing T cells is less than that required for activation of mature T cells.

Animals↗

PubMed2000

Transgenic resistance to PVY(O) associated with post-transcriptional silencing of P1 transgene is overcome by PVY(N) strains that carry highly homologous P1 sequences and recover transgene expression at infection.

Resistance to Potato virus Y (PVY) has been obtained in our previous studies through expression of the PVY P1 gene in sense or antisense orientation in potato cv. Pito. In the present study, the mechanism and strain specificity of the resistance were analyzed. Several features including low steady-state P1 mRNA expression in the resistant P1 plants indicated that resistance was based on post-transcriptional gene silencing (PTGS). Resistance was specific to PVY(O) isolates, the PVY strain group from which the P1 transgene was derived. However, according to group analyses, there was no distinguishing characteristic between the PVY(O) and PVY(N) strains P1 gene sequences. Therefore, the ability of the PVY(N) strains to overcome resistance could not be explained solely based on their P1 gene sequences. Infection with PVY(N) of the PVY(O)-resistant transgenic lines led to a recovery of expression of the P1 transgene. These data suggested that factors other than sequence homology are required in determination of the resistance specificity.

Blotting, Northern↗

PubMed1992

Conserved regulatory mechanisms of tyrosinase genes in mice and humans.

In vertebrates, melanin production is restricted to pigment cells. This cell type-specific melanogenesis is considered to involve cell type-specific expression of the tyrosinase gene. Recently, there have been several reports that sequences in the 5' flanking region of the mouse tyrosinase gene are responsible for cell type-specific expression of the transgene in mice. As the first step in the study of the evolution of the regulatory mechanisms for tyrosinase gene function in vertebrates, we constructed a fused gene, hg-Tyrs-J, which includes a 1.0-kb 5' flanking sequence of the human tyrosinase gene fused with mouse tyrosinase cDNA. By introducing the fused gene into fertilized eggs of albino mice, we obtained two mice that exhibited pigmentation in the skin and eyes and established a transgenic line from one of them. Further analyses revealed that the transgene was expressed cell type-specifically in these transgenic mice. We conclude, therefore, that the 1.0 kb 5' upstream region of the human tyrosinase gene contains conserved cis-elements essential for cell type-specific expression of the tyrosinase genes in mice and humans. Results of our study may provide a clue to elucidate the evolutionary process of regulatory mechanisms of the tyrosinase gene.

Animals↗