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At least 235 records · Page 13Linked to original sources
PubMed1999

Antisense suppression of tomato endo-1,4-beta-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening.

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.

Cellulase↗
PubMed1999

Association of the 5'HS4 sequence of the chicken beta-globin locus control region with human EF1 alpha gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits.

Whatever its field of application, animal transgenesis aims at a high level of reproducible and stable transgene expression. In the case of xenotransplantation, prevention of hyperacute rejection of grafts of animal origin requires the use of organs expressing human inhibitors of complement activation such as CD55 (DAF) and CD59. Pigs transgenic for these molecules have been produced, but with low and variable levels of expression. In order to improve cDNA expression, a vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus and the promoter and the first intron from the human EF1 alpha gene, was used to co-express human CD55 and CD59 cDNAs in transgenic rabbits. The transgenic lines with the 5'HS4 region displayed dramatically enhanced CD55 and CD59 mRNA concentrations in brain, heart, kidney, liver, lung, muscle, spleen and aortic endothelial cells in comparison with the transgenic lines without the 5'HS4 region. In the absence of the 5'HS4 region, only some of the transgenic lines displayed specific mRNAs and at low levels. Human CD55 and CD59 proteins were detectable in mononuclear cells from transgenic rabbits although at a lower level than in human mononuclear cells. On the other hand, primary aortic endothelial cells from a bi-transgenic line were very efficiently protected in vitro against human complement-dependent lysis. Transgenic rabbits harbouring the two human inhibitors of complement activation, CD55 and CD59, can therefore be used as new models in xenotransplantation. Moreover, the vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus seems appropriate not only for xenotransplantation but also for any other studies involving transgenic animals in which cDNAs have to be expressed at a high level in all cell types.

Animals↗
PubMed1997

High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.

Despite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specific actin (alpha-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal beta-actin promoter and generated transgenic zebrafish using a beta-actin-GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the beta-actin gene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene.

Actins↗
PubMed2004

Comparative studies on the type-B response regulators revealing their distinctive properties in the His-to-Asp phosphorelay signal transduction of Arabidopsis thaliana.

In Arabidopsis thaliana, a Histidine-to-Aspartate (His-->Asp) phosphorelay is involved in the signal transduction for propagation of certain stimuli, such as plant hormones. Through the phosphorelay, the type-B phospho-accepting response regulator (ARR) family members serve as DNA-binding transcriptional regulators, whose activities are most likely regulated by phosphorylation/dephosphorylation. Based on the fact that this higher plant has 11 type-B ARR family genes, we clarified the expression profiles for all of their transcripts in plants. We constructed and characterized a series of transgenic lines, each carrying a given ARR-promoter::GUS transgene. Transcripts of some type-B ARR family genes were detected more or less ubiquitously in many organs tested, while others were expressed predominantly in reproductive organs. These ARR family members were phylogenetically classified into three sub-families, the largest of which includes the well-characterized ARR1, ARR2, and ARR11. Comparative studies were conducted focusing on ARR20 and ARR21, each of which is a representative member of an uncharacterized minor sub-family. A set of transgenic lines was constructed, in each of which a C-terminal DNA-binding domain lacking the N-terminal phospho-accepting receiver of a given ARR was aberrantly overexpressed. These resulting transgenic lines, including ARR14-C-ox, ARR20-C-ox, and ARR21-C-ox, showed characteristic anomalies during development. These results are discussed with special reference to the His-->Asp phosphorelay signal transduction in A. thaliana.

Arabidopsis↗
PubMed2002

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids.

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.

Animals↗
PubMed2004

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

Agriculture↗
PubMed2004

Expression of Escherichia coli branching enzyme in caryopses of transgenic rice results in amylopectin with an increased degree of branching.

Physiochemical properties of starch are dependent on several factors including the relative abundance of amylose and amylopectin, and the degree of branching of amylopectin. Utilizing Agrobacterium-mediated transformation, a construct containing the coding region of branching enzyme of Escherichia coli, under transcriptional control of the rice (Oryza sativa L.) starch-branching enzyme promoter was introduced into rice cv. Nakdong. To enhance glgB expression, the first intron of rice starch-branching enzyme and the matrix attachment region (MAR) sequence from chicken lysozyme were included in the expression vector. Eleven independent transgenic rice plants were generated. Southern blot analysis indicated that the copy number of glgB integrated into transgenic rice varied from one to five. High-performance liquid chromatographic analysis of starch from transgenic lines revealed that amylopectin from transgenic lines exhibited greater branching than that of non-transgenic rice. The A/B1 ratio in amylopectin increased from 1.3 to 2.3 and the total branching ratio, A+B1/B-rest, increased from 6 to 12 in transgenic rice. The observed increase in the short-chain fractions with a degree of polymerization between 6 and 10 is expected to have a significant effect on retrogradation. Our study demonstrates that amylopectin branching can be altered in vivo, thus changing the physicochemical properties of starch.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1999

Mea2/Golga3 gene is disrupted in a line of transgenic mice with a reciprocal translocation between Chromosomes 5 and 19 and is responsible for a defective spermatogenesis in homozygotes.

A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation. The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization. The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis, while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis. Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604 mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes.

Animals↗
PubMed2006

Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing.

A green fluorescent protein (GFP) transgene under the control of the 35S cauliflower mosaic virus (CaMV) promoter was introduced by Agrobacterium-mediated transformation into Nicotiana benthamiana to generate fourteen transgenic lines. Homozygous lines that contained one or two copies of the transgene showed great variation of GFP expression under ultraviolet (UV) light, which allowed classification into three types of transgenic plants. Plants from more than half of the transgenic lines underwent systemic RNA silencing and produced short interfering RNA (siRNA) as young seedlings, while plants of the remaining lines developed, in a spontaneous manner, defined GFP-silenced zones on their leaves, mostly in the form of circular spots that expanded to about 4-7 mm in size. In some of the latter lines, the GFP-silenced spots remained stable, but no systemic silencing occurred. Here we characterize this phenomenon, which we term spontaneous short-range silencing (SSRS). Biochemical analysis of silenced spot tissue did not reveal detectable levels of siRNA. However, agro-infiltration with the suppressor proteins P19 of cymbidium ring spot virus (CymRSV), HC-Pro of tobacco etch virus (TEV), and crosses to a P19 transgenic line, nevertheless suggests that low concentrations of siRNA may have a functional role in the locally silenced zone. We propose that small alterations in the steady-state concentration of siRNAs and their cognate mRNA are decisive with regard to whether silencing remains local or spreads in a systemic manner.

Gene Expression Regulation, Plant↗
PubMed1994

Molecular phenotype of simian virus 40 large T antigen-induced primitive neuroectodermal tumors in four different lines of transgenic mice.

BACKGROUND: We compared the molecular phenotypes of central nervous system tumors arising in four different lines of transgenic mice (TGM) carrying the Simian virus 40 large T antigen driven by different promoters or enhancers. Two of the four lines developed primitive neuroectodermal tumors (PNETs) in the brain stem or pineal gland. A third TGM line developed retinoblastomas (a PNET-like tumor of the retina) as well as PNETs in the mesencephalon, while the fourth TGM developed retinoblastomas and adrenal pheochromocytomas. EXPERIMENTAL DESIGN: The expression of developmentally regulated polypeptides specific for the neuronal or glial lineage was examined in these PNETs using immunohistochemistry and Western blotting. RESULTS: Neoplastic cells in all of the PNETs exhibited neuronal, but no glial specific markers as evidenced by the invariable expression of synaptophysin, but no detectable glial fibrillary acidic protein or myelin basic protein. PNETs with a more differentiated neuronal phenotype expressed multiple neuronal polypeptides. The phenotypic properties of these PNETs closely resembled those found in human brain PNET biopsy samples and cell lines derived therefrom. CONCLUSIONS: We conclude that Simian virus 40 T antigen-induced PNETs in TGM exhibit the molecular phenotype of developing neurons or neuronal progenitor cells. Although many factors could influence the phenotype of these experimental PNETs (e.g., promoter, site of integration of the transgene) these PNETs appear to be suitable TGM models of human PNETs of the central nervous system.

Adrenal Gland Neoplasms↗
PubMed1990

Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate.

We describe transgenic mice that carry an antigen receptor gene minilocus comprised of germline T cell receptor (TCR) beta variable gene elements (V, D and J) linked to an immunoglobulin (Ig) C mu constant region gene with or without a DNA segment containing the Ig heavy chain transcriptional enhancer (E mu). Transgenic constructs lacking the E mu-containing segment did not undergo detectable rearrangement in any tissue of six independent transgenic lines. In contrast, transgenic constructs containing this DNA segment underwent rearrangement at high frequency in lymphoid tissues, but not other tissues, of four independent lines. Analyses of purified B and T cells, as well as B and T cell lines, from transgenic animals demonstrated that the E mu-containing segment within the construct allowed partial TCR gene assembly (D to J) in both B and T cells. However, complete TCR gene rearrangement within the construct (V to DJ) occurred only in T cells. Therefore, we have demonstrated elements that can control two separate aspects of TCR beta VDJ rearrangement within this construct. One lies within the E mu-containing DNA segment and represents a dominant, cis-acting element that initiates lymphoid cell-specific D beta to J beta rearrangement; various considerations suggest this activity may be related to that of the E mu element. The second element provides T cell-specific control of complete (V beta to DJ beta) variable region gene assembly; it correlates in activity with expression of the unrearranged V beta segment.

Animals↗
PubMed1995

Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene.

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

Base Sequence↗
PubMed2005

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines.

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.

Amides↗
PubMed1993

Susceptibility to inflammatory disease in HLA-B27 transgenic rat lines correlates with the level of B27 expression.

To investigate the role of the class I MHC molecule HLA-B27 in the spondyloarthropathies, we produced rats transgenic for HLA-B27 and human beta 2-microglobulin. Of five lines bearing > 1 copy of each transgene and showing hemizygous expression of both transgenes, two (lines 21-4H and 33-3) developed spontaneous inflammatory disease that closely resembled B27-associated human disease. Two lines, 21-4L and 25-6, remained healthy even when homozygous for the transgene locus, whereas the 21-3 line, bearing the third highest transgene copy number, developed disease similar to that of the 21-4H and 33-3 lines only when homozygous for the transgene locus. The disease-prone lines showed higher expression of B27--thymic mRNA in utero, splenic mRNA by 5 days of age, and splenic cell surface protein by the time of disease onset--than the disease-resistant lines. Disease susceptibility thus appeared to correlate with gene copy number and the quantity of B27 in lymphoid cells. The increase in the amount of B27 protein did not appear to be simply a consequence of the inflammatory disease because 1) there was no similar change in endogenous RT1 class I expression; 2) no alteration of B27 expression occurred in 21-4H rats with adjuvant-induced arthritis; 3) in rats with inflammatory disease transgenic for HLA-A2 and the 21-4H transgene locus, A2 expression was the same as in healthy rats transgenic for A2 but not B27; and 4) the transgenes in disease-prone and disease-resistant lines were equally susceptible to induction by IFN-gamma. Immunocytochemistry of the distal colon, an early site of inflammation, showed that the B27 Ag is expressed at high levels in cells of the lamina propria, but not at all in colonic epithelial cells. Taken together, the data suggest that the B27 transgene is expressed in a copy number dependent, position-independent manner in lymphoid tissue and that disease results from the expression of B27 above a critical threshold level.

Animals↗
PubMed2006

Transgenic mouse line overexpressing the cancer-specific tNOX protein has an enhanced growth and acquired drug-response phenotype.

tNOX, a novel cell surface protein related to unregulated growth and drug response of cancer cells, has been proposed as the cellular target for the anticancer action of various quinone site inhibitors with anticancer activity including the polyphenol (-)-epigallocatechin-3-gallate (EGCg). A transgenic mouse line overexpressing tNOX was generated to determine its overall growth phenotype and susceptibility to EGCg. Cultured noncancer cells lack tNOX and are unresponsive to EGCg. Overexpression of tNOX in cultured noncancer cells through transfection resulted in both enhanced growth and an acquired inhibitory response to EGCg. The tNOX transgenic mouse line was developed using a phCMV2 vector with the hemagglutinin (HA) tag. Transgenic mice exhibited both an enhanced growth rate and a response to EGCg not observed with wild-type mice. Female transgenic mice grew twice as fast as wild type, and growth was reflected in an overall increased carcass weight. Administration of EGCg in the drinking water [500 mg/kg body weight (BW)] reduced the growth rate of the transgenic mice to that of wild-type mice. The findings provide in situ validation of the hypothesis that tNOX represents a necessary and sufficient molecular target as the basis for the protective and potential cancer therapeutic benefits of EGCg.

Animals↗
PubMed2006

Physiological effects of constitutive expression of Oilseed Rape Mosaic Tobamovirus (ORMV) movement protein in Arabidopsis thaliana.

Movement proteins (MPs) are non-cell autonomous viral-encoded proteins that assist viruses in their cell-to-cell movement. The MP encoded by Tobamoviruses is the best characterized example among MPs of non-tubule-inducing plant RNA viruses. The MP of Oilseed Rape Mosaic Tobamovirus (ORMV) was transgenically expressed in Arabidopsis thaliana, ecotype RLD, under the expression of the 35S promoter from Cauliflower Mosaic Virus. Transgenic lines were obtained in sense and antisense orientations. One of the sense transgenic lines was further characterized turning out to carry one copy of the transgene inserted in the terminal region of the right arm of chromosome 1. The constitutive expression of ORMV-MP induced mild physiological effects in Arabidopsis. Plants of the transgenic line allowed a faster systemic movement of the phloem tracer carboxyfluorescein. The tracer was unloaded differentially in different flower parts, revealing differential effects of ORMV-MP on phloem unloading in sink organs. On the other hand, transgenic Arabidopsis did not show any effect on biomass partitioning or sugar availability, effects reported for equivalent transgenic solanaceous plants expressing the MP of Tobacco Mosaic Virus, another Tobamovirus. Finally, the transgenic Arabidopsis plants were susceptible to ORMV infection, although showing milder overall symptoms than non-transgenic controls. The results highlight the relevance of the specific host-virus system, in the physiological outcome of the molecular interactions established by MPs.

Arabidopsis↗
PubMed1992

Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.

Animals↗
PubMed2005

Insect-resistant transgenic Pinus radiata.

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.

Animals↗