Transgenic Research (TRANSGENIC RES)

Publications in this journal

• Article: Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model

  Yutaka Haga, Vincent J. Dominique, Shao Jun Du
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  ABSTRACT: To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.
  Transgenic Research 04/2012; 18(5):669-683.
• Article: Meeting Report: UC Davis Transgenic Animal Research Conference VII

  Louis-Marie Houdebine
  Transgenic Research 04/2012; 19(1):127-130.
• Article: Overexpression of CrtR-b2 (carotene beta hydroxylase 2) from S. lycopersicum L. differentially affects xanthophyll synthesis and accumulation in transgenic tomato plants

  Caterina D’Ambrosio, Adriana Lucia Stigliani, Giovanni Giorio
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  ABSTRACT: Plant chloroplasts are enriched in xanthophylls which participate in photosynthesis as light-absorbing pigments and as dissipaters of excess light. In comparison, chromoplasts have evolved the capacity to synthesize and store brightly coloured carotenoid pigments to give flowers and fruits the power to attract pollinators and fruit dispersers. The best performing accumulator of xanthophylls in tomato is the petal chromoplast in contrast to the fruit chromoplast which only seems able to store carotenes. We have generated genetically engineered tomato lines carrying the tomato CrtR-b2 transgene with the aim of forcing the fruit to accumulate beta-xanthophylls. Both chloroplast- and chromoplast-containing tissues of hemizygous transgenic plants were found to contain elevated xanthophyll contents as a direct consequence of the increased number of CrtR-b2 transcripts. Hemizygous transgenic leaves contained fourfold more violaxanthin than control leaves. Developing fruits were yellow instead of green since they lacked chlorophyll a, and their violaxanthin and neoxanthin contents were seven- and threefold higher, respectively, than those of the control. Ripe fruits of hemizygous transgenic plants contained free violaxanthin and significant amounts of esterified xanthophylls. Esterified xanthophylls were present also in ripe fruits of control and homozygous plants. However, in transgenic homozygous plants, we observed a reduction in transcript content in most tissues, particularly in petals, due to a post-transcriptional gene silencing process. These findings demonstrate that tomato fruit chromoplasts can accumulate xanthophylls with the same sequestration mechanism (esterification) as that exploited by chromoplasts of the tomato petal and pepper fruit. This study on transgenic plants overexpressing an important carotenoid gene (CrtR-b2) provides an interesting model for future investigations on perturbations in beta-carotene-derived xanthophyll synthesis which in turn may provide insights into the molecular mechanisms controlling carotenoid metabolism in tomato. KeywordsChlorophylls– CrtR-b2 –Esterification–Gene silencing–Transcriptional regulation–Xanthophylls
  Transgenic Research 04/2012; 20(1):47-60.
• Article: Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions

  Giovanni Giorio, Adriana Lucia Stigliani, Caterina D’Ambrosio
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  ABSTRACT: Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanum lycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was “HighCaro (HC),” a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to β-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to β-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place.
  Transgenic Research 04/2012; 16(1):15-28.
• Article: Varying phenotypes in swine versus murine transgenic models constitutively expressing the same human Sonic hedgehog transcriptional activator, K5-HGLI2ΔN

  Amy C. McCalla-Martin, Xiaoxin Chen, Keith E. Linder, Jose L. Estrada, Jorge A. Piedrahita
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  ABSTRACT: This study was undertaken to characterize the effects of constitutive expression of the hedgehog transcriptional activator, Gli2, in porcine skin. The keratinocyte-specific human transgene, K5-hGli2ΔN, was used to produce transgenic porcine lines via somatic cell nuclear transfer techniques. In mice, K5-hGli2ΔN induces epithelial downgrowths resembling basal cell carcinomas. Our porcine model also developed these basal cell carcinoma-like lesions, however gross tumor development was not appreciated. In contrast to the murine model, diffuse epidermal changes as well as susceptibility to cutaneous infections were seen in the swine model. Histologic analysis of transgenic piglets revealed generalized epidermal changes including: epidermal hyperplasia (acanthosis), elongated rete ridges, parakeratotic hyperkeratosis, epidermal neutrophilic infiltration, capillary loop dilation and hypogranulosis. By 2weeks of age, the transgenic piglets developed erythematic and edematous lesions at high contact epidermal areas and extensor surfaces of distal limb joints. Despite antibiotic treatment, these lesions progressed to a deep bacterial pyoderma and pigs died or were euthanized within weeks of birth. Non-transgenic littermates were phenotypically normal by gross and histological analysis. In summary, constitutive expression of the human hGli2ΔN in keratinocytes, results in cutaneous changes that have not been reported in the K5-hGli2ΔN murine model. These findings indicate a need for a multiple species animal model approach in order to better understand the role of Gli2 in mammalian skin. KeywordsBasal cell carcinoma-Pig-Somatic cell nuclear transfer-Gli2-Hedgehog
  Transgenic Research 04/2012; 19(5):869-887.
• Article: Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer’s disease-causing dominant mutation APPsw

  Peter M. Kragh, Anders Lade Nielsen, Juan Li, Yutao Du, Lin Lin, Mette Schmidt, Ingrid Brück Bøgh, Ida E. Holm, Jannik E. Jakobsen, Marianne G. Johansen, Stig Purup, Lars Bolund, Gábor Vajta, Arne Lund Jørgensen
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  ABSTRACT: In an effort to develop a porcine model of Alzheimer’s disease we used handmade cloning to produce seven transgenic Göttingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGFbeta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGFbeta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the Aβ peptide in the brain may develop at the age of 1–2years.
  Transgenic Research 04/2012; 18(4):545-558.
• Article: Pax1/E2a Double-Mutant Mice Develop Non-Lethal Neural Tube Defects that Resemble Human Malformations

  Paulus H.L.J. Joosten, Everardus J.J. van Zoelen, Cornelis Murre
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  ABSTRACT: Many mouse models exist for neural tube defects (NTDs), but only few of them are relevant for human patients that are born alive with spina bifida aperta. NTDs in humans show a complex inheritance, which most likely result from the involvement of a variety of predisposing genetic and environmental factors. Hints toward the identity of predisposing genetic factors for human NTDs could come from mouse studies on the development of the neural tube and spinal cord, as well as from studies on associated features of this type of diseases. Among such features is the observation that pregnancies affected by a neural tube defect frequently show changes in thymus morphology, and in both neonatal and maternal T-cell repertoire. The genes for E2a and Pax1 have both been implicated in not only paraxial mesodermal development, but also in that of the immune system. Moreover, Pax1 mutant mice have been shown to display NTDs in digenic mouse models. In the present study we have investigated the phenotype of E2a null mutant mice that are also heterozygous for the so-called undulated mutation in Pax1. Here we report that such double-mutant mice develop a non-lethal NTD that strongly resembles the classic human NTD: spina bifida aperta, associated with defects of the axial skeleton, immune system and urinary tract.
  Transgenic Research 04/2012; 14(6):983-987.
• Article: Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpinXooc-encoding hrf2 Gene

  Rong Huo, Yu Wang, Ling-Li Ma, Jun-Qing Qiao, Min Shao, Xue-Wen Gao
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  ABSTRACT: hrf2 gene is a member of the harpin-encoding gene family of rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola. In our previous studies, we observed that harpinXooc could elicit hypersensitive cell death in non-host plants, induce disease and insect resistance in plants, and enhance plant growth. In this study, the rapeseed cultivar, Yangyou 4, was genetically engineered via Agrobacterium-mediated transformation to express the hrf2 gene. Polymerase chain reaction (PCR) and southern blot analyses of T1 generation of transgenic rapeseed revealed stable integration and expression of the inserted gene hrf2. In addition, the resistance to Sclerotinia sclerotiorum was greatly enhanced. A comparison between agronomic characters of transgenic and control lines displayed significant differences in terms of plant height, stem width, number of pods per plant, number of seeds per pod, 1,000-seed weight, and seed yield per plant. Among lines with resistance to S. sclerotiorum, T11 had improved agronomic traits compared with controls with a 22.7% seed yield increase. These results suggest that the introduction of the hrf2 gene into rapeseed can be an effective strategy for enhancing resistance to S. sclerotiorum. KeywordsTransgenic rapeseed- hrf2 gene-Inheritance-Resistance-Agronomic performance
  Transgenic Research 04/2012; 19(5):841-847.
• Article: ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome

  James G. Thomson, Yuan-Yeu Yau, Robert Blanvillain, Dawn Chiniquy, Roger Thilmony, David W. Ow
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  ABSTRACT: The small serine resolvase ParA from bacterial plasmids RK2 and RP4 catalyzes the recombination of two identical 133bp recombination sites known as MRS. Previously, we reported that ParA is active in the fission yeast Schizosaccharomyces pombe. In this work, the parA recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis lines harboring a chromosomally integrated MRS-flanked target. The ParA recombinase excised the MRS-flanked DNA and the excision event was detected in subsequent generations in the absence of ParA, indicating germinal transmission of the excision event. The precise site-specific deletion by the ParA recombination system in planta demonstrates that the ParA recombinase can be used to remove transgenic DNA, such as selectable markers or other introduced transgenes that are no longer desired in the final product. KeywordsCre-lox -Marker excision-Genetic engineering- OXS3 -Site-specific recombination
  Transgenic Research 04/2012; 18(2):237-248.
• Article: Commentary on Houdebine's review

  Henryk Lubon, Carol Palmer
  Transgenic Research 04/2012; 9(4):301-304.
• Article: Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

  Kazuhito Izukuri, Kenji Suzuki, Nobuyuki Yajima, Shigeyuki Ozawa, Shin Ito, Eiro Kubota, Ryu-Ichiro Hata
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  ABSTRACT: We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11ng/ml plasma, respectively) than normal blood level (0.9ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects. KeywordsChemokine CXCL14/BRAK-Tumor suppression-Lewis lung carcinoma cells-Transgenic mouse-Tumor therapy
  Transgenic Research 04/2012; 19(6):1109-1117.
• Article: Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

  Daniel F. Carlson, Aron M. Geurts, John R. Garbe, Chang-Won Park, Artur Rangel-Filho, Scott M. O’Grady, Howard J. Jacob, Clifford J. Steer, David A. Largaespada, Scott C. Fahrenkrug
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  ABSTRACT: Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline. KeywordsSleeping Beauty–Transposon–Transgenesis–Mouse–Rat–Methylation
  Transgenic Research 04/2012; 20(1):29-45.
• Article: Generation of stable Xenopuslaevis transgenic lines expressing a transgene controlled by weak promoters

  Anne L’hostis-Guidet, Gaëlle Recher, Brigitte Guillet, Abdulrahim Al-Mohammad, Pascal Coumailleau, François Tiaho, Daniel Boujard, Thierry Madigou
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  ABSTRACT: Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.
  Transgenic Research 04/2012; 18(5):815-827.