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At least 721 records · Page 40Linked to original sources
PubMed2003

Sry expression level and protein isoform differences play a role in abnormal testis development in C57BL/6J mice carrying certain Sry alleles.

Transfer of certain Mus domesticus-derived Y chromosomes (Sry(DOM) alleles, e.g., Sry(POS) and Sry(AKR)) onto the C57BL/6J (B6) mouse strain causes abnormal gonad development due to an aberrant interaction between the Sry(DOM) allele and the B6-derived autosomal (tda) genes. For example, B6 XY(POS) fetuses develop ovaries and ovotestes and B6 XY(AKR) fetuses have delayed testis cord development. To test whether abnormal testis development is caused by insufficient Sry(DOM) expression, two approaches were used. First, gonad development and relative Sry expression levels were examined in fetal gonads from two strains of B6 mice that contained a single M. domesticus-derived and a single M. musculus-derived Sry allele (B6-Y(POS,RIII) and B6-Y(AKR,RIII)). In both cases, presence of the M. musculus Sry(RIII) allele corrected abnormal testis development. On the B6 background, Sry(POS) was expressed at about half the level of Sry(RIII) whereas Sry(AKR) and Sry(RIII) were equally expressed. On an F(1) hybrid background, both Sry(POS) and Sry(RIII) expression increased, but Sry(POS) expression increased to a greater extent. Second, sexual development and Sry expression levels were determined in XX mice carrying a transgene expressing Sry(POS) controlled by POS-derived or MUS-derived regulatory regions. In both cases one B6 transgenic line was recovered in which XX transgenic mice developed only testicular tissue but cord development was delayed despite normal Sry transcriptional initiation and overexpression. For three transgenes where B6 XX transgenic mice developed as females, hermaphrodites, or males, the percentage of XX transgenic males increased on an F(1) background. For the one transgene examined, Sry expression increased on an F(1) background. These results support a model in which delayed testis development is caused by the presence of particular DOM SRY protein isoforms and this, combined with insufficient Sry expression, causes sex reversal. These results also indicate that at least one tda gene regulates Sry expression, possibly by directly binding to Sry regulatory regions.

Animals↗
PubMed2003

Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice.

The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx(a), which produces a large amount of amylose, and Wx(b), which produces a smaller amount of amylose because of the mutation at the 5' splice site of intron 1. Wx(b) is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx(a) cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx(a) transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.

Amylose↗
PubMed1999

Transformation of tobacco and potato with cDNA encoding the full-length genome of potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication.

A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.

DNA, Complementary↗
PubMed2003

Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance.

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2006

Stable skin-specific overexpression of human CTLA4-Ig in transgenic mice through seven generations.

Skin graft rejection is a typical cellular immune response, mainly mediated by T cells. Cytotoxic T lymphocyte associated antigen 4-immunoglobin (CTLA4-Ig) extends graft survival by blocking the T cell co-stimulation pathway and inhibiting T cell activation. To investigate the efficacy of CTLA4-Ig in prolonging skin graft survival, human CTLA4-Ig (hCTLA4-Ig) was engineered to overexpress in mouse skin by transgenesis using the K14 promoter. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay indicated that the expression of CTLA4-Ig remained skin-specific and relatively constant compared to the internal control protein, AKT, through seven generations. The presence and concentration of the hCTLA4-Ig protein in transgenic mouse sera was determined by enzyme-linked immunosorbent assay (ELISA), and the results indicated that the serum CTLA4-Ig concentration also remained constant through generations. Survival of transgenic mouse skins grafted onto rat wounds was remarkably prolonged compared to that of wild-type skins from the same mouse strain, and remained comparable among all seven generations. This suggested that the bioactive hCTLA4-Ig protein was stably expressed in transgenical mice through at least seven generations, which was consistent with the stable skin-specific CTLA4-Ig expression. The results demonstrated that the transgenic expression of hCTLA4-Ig in skin driven by the K14 promoter remained constant through generations, and a transgenic line can be established to provide transgenic skin with extended survival reproducibly.

Abatacept↗
PubMed1991

The mitochondrial uncoupling protein gene in brown fat: correlation between DNase I hypersensitivity and expression in transgenic mice.

The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I-hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo.

Adipose Tissue, Brown↗
PubMed2004

New insight into progenitor/stem cells in dental pulp using Col1a1-GFP transgenes.

In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application in tissue engineering. These advances provide a promising future for tooth replacement/regeneration. Essential for this approach is the identification of donor stem cells for various components of the teeth. Our studies show that pOBCol3.6GFPtpz and pOBCol2.3GFPemd transgenic animals provide a unique model to gain insight into stem cells in the dental pulp. Our in vivo studies of the developing teeth of these transgenic lines show both Col1a1-GFP transgenes are expressed in functional and fully differentiated odontoblasts. The patterns of expression of Col1a1-GFP transgenes during odontoblast differentiation correlates with the expression of DSPP. In the developing craniofacial bones both Col1a1-GFP transgenes are also expressed in osteoblasts and osteocytes of alveolar and calvarial bones. In the alveolar bones, the expression of Col1a1-GFP in osteocytes correlates with the expression of DMP1. Col1a1-3.6-GFP is expressed in the entire layer of the periosteum and in suture mesenchyme containing osteoprogenitor cells. On the other hand, Col1a1-2.3- GFP expression was limited to the osteoblastic layer of the periosteum and was not detected in the fibroblastic layer of the periosteum or in the suture mesenchyme. These observations indicate that Col1a1-3.6-GFP and Col1a1-2.3-GFP transgenes identify different subpopulations of cells during intramembranous ossification. By using the coronal portion of dental pulps isolated from postnatal transgenic mice our observations also provide direct evidence that the dental pulp contains progenitor/stem cells capable of giving rise to a new generation of odontoblast-like cells, as well as osteoblast-like cells.

Ameloblasts↗
PubMed1999

Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia.

We generated transgenic mice that carry 4.6 kb of the mouse galanin gene fused to 2.5 kb of the rat PRL promoter. In all transgenic lines that carried and transmitted the transgene, there were significant increases in galanin messenger RNA and peptide levels in the anterior pituitary in both male and female transgenic mice, and the elevation of galanin was restricted to the anterior lobe. Furthermore, galanin release from pituitary cells in vitro of both male and female transgenic mice was dramatically increased compared with that in control mice. At 2-4 months of age, pituitary PRL contents in female transgenic mice were increased compared with those in normal controls. Moreover, PRL messenger RNA levels were increased in female transgenic mice. However, plasma levels of PRL in female transgenic mice were not significantly higher until 6 months of age. By 11 months of age, cell numbers in the anterior pituitary were increased in female, but not male, transgenic mice. The percentage of lactotrophs in female transgenic mice as well as PRL gene expression per cell were significantly higher. No differences were detected in PRL content, gene expression, or release between normal and transgenic male mice. Six weeks of estrogen treatment significantly increased anterior pituitary weights and PRL secretion in male transgenic mice compared with that in normal male mice. In addition, anterior pituitary weights and PRL secretion were decreased in female transgenic mice compared with controls 6 weeks after ovariectomy. We conclude that overexpression of galanin in lactotrophs stimulates PRL synthesis and secretion and acts as a growth factor resulting in the formation of pituitary hyperplasia and hyperprolactinemia. Furthermore, estrogen appears critical for these galanin-mediated events.

Aging↗
PubMed2006

Fitness benefits of systemic acquired resistance during Hyaloperonospora parasitica infection in Arabidopsis thaliana.

We investigated the fitness benefits of systemic acquired resistance (SAR) in Arabidopsis thaliana using a mutational and transformational genetic approach. Genetic lines were designed to differ in the genes determining resistance signaling in a common genetic background. Two mutant lines (cpr1 and cpr5) constitutively activate SAR at different points in SAR signaling, and one mutant line (npr1) has impaired SAR. The transgenic line (NPR1-H) has enhanced resistance when SAR is activated, but SAR is still inducible similarly to wild type. The fitness benefits were also investigated under two nutrient levels to test theories that preventing pathogen damage and realized resistance benefits may be affected by nutrient availability. Under low-nutrient conditions and treatment with the pathogenic oomycete, Hyaloperonospora parasitica, wild type had a higher fitness than the mutant that could not activate SAR, demonstrating that normal inducible SAR is beneficial in these conditions; this result, however, was not found under high-nutrient conditions. The mutants with constitutive SAR all failed to show a fitness benefit in comparison to wild type under a H. parasitica pathogen treatment, suggesting that SAR is induced to prevent an excessive fitness cost.

Arabidopsis↗
PubMed1997

Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

To develop a syngeneic transplantable system to study immunotherapeutic approaches for the treatment of prostate cancer, three cell lines were established from a heterogeneous 32 week tumor of the transgenic adenocarcinoma mouse prostate (TRAMP) model. TRAMP is a transgenic line of C57BL/6 mice harboring a construct comprised of the minimal -426/+28 rat probasin promoter driving prostate-specific epithelial expression of the SV40 large T antigen. TRAMP males develop histological prostatic intraepithelial neoplasia by 8-12 weeks of age that progress to adenocarcinoma with distant metastases by 24-30 weeks of age. The three cell lines (TRAMP-C1, TRAMP-C2, and TRAMP-C3) express cytokeratin, E-cadherin, and androgen receptor by immunohistochemical analysis and do not appear to have a mutated p53. Although TRAMP-C1 and TRAMP-C2 are tumorigenic when grafted into syngeneic C57BL/6 hosts, TRAMP-C3 grows readily in vitro but does not form tumors. The T antigen oncoprotein is not expressed by the cell lines in vitro or in vivo. The rationale for establishing multiple cell lines was to isolate cells representing various stages of cellular transformation and progression to androgen-independent metastatic disease that could be manipulated in vitro and, in combination with the TRAMP model, provide a system to investigate therapeutic interventions, such as immunotherapy prior to clinical trials.

Adenocarcinoma↗
PubMed2005

Challenges in creating a vaccine to prevent hepatitis E.

Recombinant hepatitis E virus capsid protein (HEV CP) assembles orally immunogenic virus-like particles (VLP) when expressed in an insect cell system. We used plant expression cassettes, pHEV101 and pHEV110, for transformation of potato to express HEV CP, and 10 independent transgenic lines of HEV101 and 6 lines of HEV110 were obtained. ELISA for HEV CP was performed on tuber extracts. Accumulation of HEV CP in tubers varied from about 5 to 30 microg/g fresh tuber depending on the transgenic plant line. We further compared the expression levels with the yield of tubers for each line. Tuber yield varied less than expression levels, and ranged from about 600 to 1000 g per pot. Although Western blot showed that apparently intact HEV CP accumulated, we observed very limited assembly of virus-like particles in potato tubers. Oral immunization of mice with transgenic potatoes failed to elicit detectable anti-CP antibody response in serum, suggesting that VLP assembly is a key factor in orally delivered HEV CP vaccines.

Animals↗
PubMed2000

Mice transgenic for monocyte-tropic HIV type 1 produce infectious virus and display plasma viremia: a new in vivo system for studying the postintegration phase of HIV replication.

To generate an in vivo system for investigating the postintegration phase of HIV-1 replication, mouse lines transgenic for a full-length infectious proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1JR-CSF, were constructed. Leukocytes from two independent JR-CSF transgenic mouse lines produced HIV-1 that infected human PBMCs. Plasma viremia was detected in these mice at levels (mean, >60,000 HIV RNA copies/ml) comparable to those reported for HIV-1-infected individuals. The levels of HIV RNA in these mice increased several-fold after either treatment with the superantigen Staphylococcus enterotoxin B or infection with Mycobacterium tuberculosis. Thus, a provirus encoding a monocyte-tropic HIV-1 strain under the control of its LTR expressed as a transgene in mice can proceed through the postintegration replication phase and produce infectious virus. In addition, the presence of plasma viremia that can be monitored by measuring plasma HIV-1 RNA levels permits these mice to be used to study the impact of different interventions on modulating in vivo HIV-1 production. Therefore, these mice provide a novel manipulable system to investigate the in vivo regulation of HIV-1 production by factors that activate the immune system. Furthermore, this murine system should be useful in delineating the role of human-specific factors in modulating HIV-1 replication and investigating the in vivo therapeutic efficacy of agents that target the postintegration stages of HIV-1 replication.

Animals↗
PubMed2001

Cre-mediated transgene activation in the developing and adult mouse brain.

The neuron-specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre-mediated transgene activation was determined by PCR and/or X-gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE-Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole-mount X-gal staining of 9.5 dpc embryos indicated Cre-mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5-13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre-mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE-Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain.

Animals↗
PubMed1993

Chicken neuronal acetylcholine receptor alpha 2-subunit gene exhibits neuron-specific expression in the brain and spinal cord of transgenic mice.

Transgenic mice carrying the complete structural gene of the alpha 2 subunit of the chicken neuronal nicotinic acetylcholine receptor (nAChR) and 7 kilobase pairs (kbp) of 5' upstream and 3 kbp of 3' downstream sequences have been generated. The transgene was stably integrated in transgenic lines and transmitted to their progeny. Avian transgene expression was predominant in the central nervous system as detected by specific alpha 2-subunit cDNA amplification. Moreover, in at least two independent mouse lines, its expression appeared to be neuron-specific and reproducibly restricted to subregions in the brain and spinal cord, as revealed by in situ hybridization histochemistry. Most cranial motor nuclei were positive, and several of the alpha 2-subunit transgene-expressing structures corresponded to cholinergic areas in rodents. This study reveals that regulatory mechanisms giving rise to neuronal-specific gene expression have been conserved at least in part between birds and mammals.

Animals↗
PubMed2002

Cre/loxP recombination-activated neuronal markers in mouse neocortex and hippocampus.

A new strategy for visualizing neuronal cell morphology of mouse brain based on Cre/loxP recombination-activated gene expression is described. A "reporter" transgenic line was generated which expressed a fusion gene encoding a dendrite-targeted green fluorescent protein (MAP2-GFP) upon deletion of a transcription/translation STOP (transcription and translation stop signal) cassette. Cre transgenic "deleter" lines were established that activated reporter gene expression at various frequencies in pyramidal neurons in the forebrain. A deleter line was identified which activated a MAP2-GFP reporter gene at very low frequency (less than 0.1% of pyramidal neurons) and allowed the visualization of dendritic structures of individual neocortical and hippocampal pyramidal neurons. In addition, vertical "columns" of pyramidal neurons in the neocortex were labeled in these mice. In a second deleter line, a MAP2-GFP reporter gene was selectively activated in pyramidal neurons of the CA-1 subregion of the hippocampus in young mice. With its combinatorial property, this binary recombination-activated neuronal marker system should facilitate the study of detailed morphology, connectivity, and plasticity of defined classes of live neurons in vitro and in vivo.

Animals↗
PubMed2006

Accurate determination of zygosity in transgenic rice by real-time PCR does not require standard curves or efficiency correction.

A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCycler system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCycler software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.

Genotype↗
PubMed2005

Molecular characterization of grapevine plants transformed with GFLV resistance genes: I.

The Grapevine FanLeaf Virus-Coat Protein (GFLV CP) gene was inserted through Agrobacterium-mediated transformation in Vitis vinifera "Nebbiolo", "Lumassina" and "Blaufränkisch". Two plasmids were used: pGA-CP+ (full-length GFLV CP gene with an introduced start codon) and pGA-AS (same gene in antisense orientation). Forty-three transgenic lines were regenerated. As several lines in Southern blots share same hybridization patterns, eight independent line groups resulted for "Nebbiolo", one for "Lumassina", and two for "Blaufränkisch". Inserted T-DNA copies ranged from one to three; one line probably contains an incomplete copy of T-DNA. Except for one "Nebbiolo" line, no evidence for methylation of the transgene at cytosine residues was found by Southern analyses. Specific mRNA was present at variable expression levels; some lines accumulated the coat protein while in others the protein was not detectable by ELISA.

Blotting, Southern↗
PubMed1990

A promoter that drives transgene expression in cerebellar Purkinje and retinal bipolar neurons.

A genomic clone encoding the Purkinje cell-specific L7 protein has been isolated and utilized to drive the expression of beta-galactosidase in mice. Three independent transgenic lines, germ line transformed with an L7-beta-galactosidase fusion gene, exhibit beta-galactosidase expression in both cerebellar Purkinje cells and retinal bipolar neurons. This distribution is the same as that previously determined for the L7 protein by immunohistochemistry. The transgenic murine lines can be used to obtain populations of marked Purkinje and bipolar neurons. Similar L7 promoter constructs can be used to express other foreign genes specifically in these two classes of neurons.

Animals↗