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1
Marnis, Huria, Bambang Iswanto, Selny Febrida, Imron Imron und Raden Roro Sri Pudji Sinarni Dewi. „TRANSMISI GEN PhGH DAN PERFORMA PERTUMBUHAN IKAN LELE AFRIKA (Clarias gariepinus) TRANSGENIK GENERASI KETIGA“. Jurnal Riset Akuakultur 11, Nr. 3 (11.01.2017): 225. http://dx.doi.org/10.15578/jra.11.3.2016.225-234.
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Penelitian ini bertujuan untuk mengevaluasi transmisi transgen PhGH dan performa ikan lele Afrika transgenik generasi ketiga (F3) yang meliputi pertumbuhan, rasio efisiensi konversi pakan, konsentrasi hormon pertumbuhan dan hormon IGF-I. Ikan lele transgenik F3 telah diproduksi dengan meyilangkan ikan lele transgenik F2 dengan non-transgenik. Deteksi transgen (PhGH) dilakukan menggunakan metode PCR. Analisis hormon pertumbuhan dan hormon insuline-like growth factor (IGF-I) menggunakan sampel serum darah dan metode enzyme linked immunosorbent assay (ELISA). Hasil penelitian menunjukkan bahwa ikan lele transgenik F3 yang digunakan pada pengujian ini terdeteksi positif membawa transgen dengan ukuran fragmen gen sebesar 1.500 bp. Transmisi transgen dari induk F2 ke F3 berkisar 0-75%. Pertumbuhan bobot populasi ikan lele transgenik F3 lebih tinggi 51,26% dibandingkan dengan ikan lele non-transgenik (P<0,05). Pertumbuhan bobot populasi ikan transgenik mencapai 484±60,3 g, sedangkan pertumbuhan bobot ikan non-transgenik 319,98±65,3 g. Nilai rasio konversi pakan ikan lele transgenik F3 sebesar 0,89 sedangkan non-transgenik 1,30. Hal ini menunjukkan bahwa efisiensi pakan ikan lele transgenik F3 lebih tinggi dibandingkan dengan ikan non-transgenik (P<0,05). Ikan lele transgenik mempunyai konsentrasi hormon pertumbuhan (5,67±2,65 ng/mL) yang lebih tinggi (P<0,05) jika dibandingkan dengan ikan lele non-transgenik (3,00±1,41 ng/mL). Ikan lele transgenik juga memiliki kandungan hormon IGF-I (6,63±0,11 ng/mL) lebih tinggi (P<0,05) dibandingkan dengan ikan lele non-transgenik (5,38±0,63 ng/mL). Tingginya konsentrasi hormon pertumbuhan dan hormon IGF-I dapat mewakili performa pertumbuhan dan efisiensi penggunaan pakan pada ikan lele transgenik.The aim of this study was to determine the transmission of a transgene (PhGH) and to evaluate the performance of F3 transgenic African catfish, such as body weight, feed conversion ratio (FCR), growth hormone and IGF-I hormone profile. F3 transgenic were produced by mating F2 transgenic with non-transgenic fish. Detection of transgene was performed using PCR method. Analysis of the growth hormone and the insulin-like growth factor-I (IGF-I) hormone were conducted by enzyme linked immunosorbent assay (ELISA) method using serum samples. The results showed that the transgenic catfish F3 was positive carrying the transgene 1.500 bp. Transgene transmission from F2 to F3 ranged zero to 75%. The performance of F3 transgenic African catfish was significantly better 51.26% than that the non-transgenic (P<0.05). The body weight of transgenic population (484±255.25 g) was higher than that non-transgenic (319.98±165.27 g). FCR of transgenic fish (0.89) was lower than that non-transgenic (1.30). The growth hormone level of transgenic (5.67±2.65 ng/mL) was higher than that non-transgenic (3.00±1.41 ng/mL), IGF-I hormone level of F3 transgenic (6.63±0.11 ng/mL) was also higher than that non-transgenic (5.38±0.63 ng/mL). High level of growth hormone and IGF-I hormone represented both growth performance and efficiency of feed utilization of transgenic African catfish.
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Marnis, Huria, Bambang Iswanto, Romy Suprapto, Imron Imron und Raden Roro Sri Pudji Sinarni Dewi. „IDENTIFIKASI ZIGOSITAS IKAN LELE (Clarias gariepinus) TRANSGENIK F-2 YANG MEMBAWA GEN HORMON (PhGH) DENGAN MENGGUNAKAN METODE REALTIME-qPCR“. Jurnal Riset Akuakultur 11, Nr. 1 (14.11.2016): 39. http://dx.doi.org/10.15578/jra.11.1.2016.39-46.
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Produktivitas ikan budidaya dapat ditingkatkan melalui teknologi transgenesis. Populasi ikan lele transgenik cepat tumbuh telah dihasilkan dan karakter biologisnya telah diketahui. Namun informasi zigositas ikan lele transgenik perlu ditelaah lebih lanjut. Penelitian ini bertujuan untuk mengidentifikasi zigositas ikan lele transgenik F-2. Zigositas ikan lele transgenik diidentifikasi dengan menggunakan metode real-time qPCR (RT-qPCR) dan uji progeni. Identifikasi zigositas melalui uji progeni, dilakukan dengan mendeteksi transgen (PhGH) pada individu-individu F-3 hasil persilangan transgenik F-2 dengan non-transgenik. Hasil penelitian menunjukkan bahwa zigositas pada ikan lele transgenik F-2 dapat diidentifikasi dengan menggunakan metode RT-qPCR. Semua ikan transgenik F-2 adalah heterozigot, dengan nilai 2-Ct yang hampir sama tiap individu F-2, yaitu berkisar 0,80-0,99. Identifikasi zigositas dengan metode RT-qPCR menunjukkan hasil yang sama dengan uji progeni, semua transgenik F-2 tidak menghasilkan 100% anakan F-3 positif transgen. Pada uji progeni, transmisi transgen pada penelitian ini tidak mengikuti hukum segregasi Mendel, dengan kisaran sebesar 5%-40%.Fish farming productivity can be increased by transgenesis technology. On the previous study, transgenic African catfish population fast growing has been produced and its biological characters has been known. However information of transgenic zygosity of catfish should be examined. The aim of this study was to identify the zygosity of F-2 transgenic African catfish. The zygosity of F-2 transgenic was identified by real time-qPCR (RT-qPCR) method and progeny test. Further, identification of zygosity F-2 transgenic African catfish was confirmed by progeny test, while F-2 transgenic African catfish was mated with non-transgenic. Identification of zygosity F-2 transgenic was conducted by detection PhGH gene (transgene) in F-3 transgenic African catfish population. Transgene transmission was evaluated by PCR method. The result showed that the zygosity F-2 transgenic African catfish could be identified by RT-qPCR method. All F-2 transgenic African catfish were heterozygous, where as the 2-Ct value was almost same for all individual, which ranges from 0.80 to 0.99. The result of zygosity identification using RT-qPCR method was as same as that of progeny test. In the progeny test, transgene transmission in this study was non-Mendelian segregation, with ranges of 5%-40%.
3
Altmann, Michael, und Klaus Ammann. „Gentechnologie im gesellschaftlichen Spannungsfeld: Züchtung transgener Kulturpflanzen“. GAIA - Ecological Perspectives for Science and Society 1, Nr. 4 (01.07.1992): 204–13. http://dx.doi.org/10.14512/gaia.1.4.5.
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Bauer, I., F. Meuser und P. Brokamp. „Zöliakieforschung: Herstellung von Weizenproteinen mit transgener Hefe“. Chemie Ingenieur Technik 77, Nr. 8 (August 2005): 1181. http://dx.doi.org/10.1002/cite.200590330.
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Blum. „Transgene Pflanzen“. Praxis 91, Nr. 49 (01.12.2002): 2119–24. http://dx.doi.org/10.1024/0369-8394.91.49.2119.
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Die molekularbiologische Forschung und rekombinante DNA-Technologie hat in den letzten Jahren nicht nur die Diagnostik, Therapie und Prävention in der Humanmedizin revolutioniert, sondern auch zu enormen Entwicklungen auf dem Gebiet der Pflanzenbiotechnologie geführt. So ist es heute möglich, eine Vielzahl von Antigenen, Antikörpern und Medikamenten in transgenen Pflanzen herzustellen. Neben den Grundlagen der molekularen Pflanzengenetik und der Herstellung transgener Pflanzen werden in dieser Übersicht der aktuelle Stand der Herstellung biomedizinisch relevanter «Pflanzenprodukte», deren weitere Perspektiven, aber auch die potenziellen Probleme aufgezeigt. Es ist eine der besonderen Herausforderungen der modernen Medizin, auch an den klinisch tätigen Mediziner in einem zunehmend informierten Umfeld, die neuen biotechnologischen Verfahren zu kennen und zu verstehen, um ihren aktuellen und zukünftigen Stellenwert für Klinik und Praxis qualifiziert beurteilen zu können.
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Feldmann, S. D., S. Brandes, E. Pfeilstetter, A. Matzk und J. Schiemann. „Begleituntersuchungen des Landes Niedersachsen zur Freisetzung transgener, herbizidresistenter Rapspflanzen“. Bundesgesundheitsblatt 41, Nr. 12 (Dezember 1998): 536–42. http://dx.doi.org/10.1007/bf03044273.
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7
W�hrmann, Klaus. „Ein Beitrag zur Diskussion �ber die Freilassung transgener Organismen“. Naturwissenschaften 78, Nr. 5 (Mai 1991): 209–14. http://dx.doi.org/10.1007/bf01136081.
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8
Wohrmann, Klaus. „Ein Beitrag zur Diskussion �ber die Freilassung transgener Organismen“. Naturwissenschaften 78, Nr. 4 (April 1991): 154–57. http://dx.doi.org/10.1007/bf01136201.
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9
Müller, Andreas J., und Dieter Mettin. „Ergebnisse und Probleme bei der züchterischen Nutzung transgener Pflanzen“. Die Kulturpflanze 36, Nr. 1 (März 1988): 275–88. http://dx.doi.org/10.1007/bf01986965.
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10
Syahputra, Khairul, Flandrianto Sih Palimirmo und Yogi Himawan. „TRANSMISI GEN krt-GP11 DAN PERFORMA KETAHANAN IKAN MAS (Cyprinus carpio) TRANSGENIK F-2 TERHADAP INFEKSI KHV“. Jurnal Riset Akuakultur 11, Nr. 4 (17.01.2017): 297. http://dx.doi.org/10.15578/jra.11.4.2016.297-305.
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Pembentukan ikan mas transgenik merupakan salah satu program penelitian di Balai Penelitian Pemuliaan Ikan, Sukamandi dalam rangka menghasilkan varietas unggul ikan mas tahan infeksi KHV (Koi herpesvirus). Pada tahun 2015 telah dilakukan pembentukan ikan mas transgenik tahan KHV generasi F-2. Penelitian ini bertujuan untuk mengevaluasi transmisi gen krt-GP11, ketahanan ikan mas transgenik F-2 terhadap infeksi KHV, keberadaan marka Cyca-DAB1*05 tahan KHV pada populasi ikan mas transgenik F-2. Ikan mas transgenik F-2 dihasilkan dengan memijahkan ikan mas transgenik F-1 jantan dengan betina non-transgenik. Pengujian transmisi transgen dan deteksi marka ketahanan KHV pada transgenik F-2 dilakukan dengan metode PCR menggunakan primer spesifik untuk transgen krt-GP11 dan gen Cyca-DAB1*05. Evaluasi ketahanan ikan mas transgenik F-2 terhadap infeksi KHV dilakukan dengan uji tantang secara kohabitasi. Hasil penelitian menunjukkan bahwa transmisi gen krt-GP11 pada keturunan F-2 memiliki persentase yang relatif rendah yaitu sebesar 0%-2%. Ikan mas transgenik F-2 memiliki ketahanan relatif baik terhadap KHV dengan sintasan uji tantang sebesar 90% dan tidak berbeda nyata dengan ikan mas pembanding atau non-transgenik (P>0,05). Tingginya pesentase keberadaan marka Cyca-DAB1*05 pada populasi transgenik berperan pada ketahanan ikan mas transgenik terhadap infeksi KHV.Creating of transgenic common carp is one of the breeding programs in Research Institute for Fish Breeding for producing a superior strain of common carp resistant to KHV(Koi herpesvirus). Since 2015, the creation of common carp transgenic has been conducted to produce F2 population resistant to KHV. This study was aimed to evaluate the transmission of krt-GP11 gen,the resistantce of F2 transgenic common carp against to KHV infection, and the existence of Cyca-DAB1*05 marker resistant to KHV in F2 transgenic population. F2 transgenic population has been produced by mating F1 transgenic male with non transgenic female. Transgene transmission and the existence of marker resistant to KHV in F2 transgenic population were evaluated by PCR method using specific primer to krt-GP11 gene and Cyca-DAB1*05 gene, respectively. The resistance of F2 transgenic population againstto KHV infection was evaluated by challenge test using cohabitation method. The result showed that transmission of krt-GP11 gene in F2transgenic population was relatively low with percentage of 0-2%. The resistance of F2 transgenic common carp against to KHV was relatively high with survival rate of 90% and was not significantly different from non transgenic (p>0.05). High percentage of transgenic population having Cyca-DAB1*05 marker had a role in resistance of transgenic population against KHV infection.
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Wolf-van Bürck, L., M. Schuster, A. Bähr, N. Klymiuk, V. Weisbach, E. Wolf und J. Seissler. „Langzeitüberleben LEA29Y transgener Schweineinselzellen in humanisierten Mausmodellen ohne systemische Immunsuppression“. Diabetologie und Stoffwechsel 12, S 01 (05.05.2017): S1—S84. http://dx.doi.org/10.1055/s-0037-1601620.
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Brumme, M. „Albrecht Müller: Ethische Aspekte der Erzeugung und Haltung transgener Nutztiere“. TATuP - Zeitschrift für Technikfolgenabschätzung in Theorie und Praxis 5, Nr. 3 (01.09.1996): 59–60. http://dx.doi.org/10.14512/tatup.5.3.59.
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13
Lanchier, N., und C. Neuhauser. „Voter Model and Biased Voter Model in Heterogeneous Environments“. Journal of Applied Probability 44, Nr. 03 (September 2007): 770–87. http://dx.doi.org/10.1017/s0021900200003429.
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With the rapid adoption of transgenic crops, gene flow from transgenic crops to wild relatives through pollen dispersal is of significant concern and warrants both empirical and theoretical studies to assess the risk of introduction of transgenes into wild populations. We propose to use the (biased) voter model in a heterogeneous environment to investigate the effects of recurrent gene flow from transgenic crop to wild relatives. The model is defined on the d-dimensional integer lattice that is divided into two parts, Δ and Z d \ Δ. Individuals carrying the transgene and individuals carrying the wild type gene compete according to the evolution rules of a (biased) voter model on Z d \ Δ, while the process is conditioned to have only individuals carrying the transgene on Δ. Our main findings suggest that unless transgenes confer increased fitness in wild relatives, introgression of transgenes into populations of wild plants is slow and may even be reversible without intervention. Our study also addresses the effects of different spatial planting patterns of transgenic crops on the rate of introgression.
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Lanchier, N., und C. Neuhauser. „Voter Model and Biased Voter Model in Heterogeneous Environments“. Journal of Applied Probability 44, Nr. 3 (September 2007): 770–87. http://dx.doi.org/10.1239/jap/1189717544.
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With the rapid adoption of transgenic crops, gene flow from transgenic crops to wild relatives through pollen dispersal is of significant concern and warrants both empirical and theoretical studies to assess the risk of introduction of transgenes into wild populations. We propose to use the (biased) voter model in a heterogeneous environment to investigate the effects of recurrent gene flow from transgenic crop to wild relatives. The model is defined on the d-dimensional integer lattice that is divided into two parts, Δ and Zd \ Δ. Individuals carrying the transgene and individuals carrying the wild type gene compete according to the evolution rules of a (biased) voter model on Zd \ Δ, while the process is conditioned to have only individuals carrying the transgene on Δ. Our main findings suggest that unless transgenes confer increased fitness in wild relatives, introgression of transgenes into populations of wild plants is slow and may even be reversible without intervention. Our study also addresses the effects of different spatial planting patterns of transgenic crops on the rate of introgression.
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Fladung, Matthias, und Hans H�nicka. „Erzeugung transgener steriler Zitterpappeln zur Verhinderung eines vertikalen Gentransfers in forstliche �kosysteme“. Gesunde Pflanzen 56, Nr. 7-8 (28.10.2004): 195–200. http://dx.doi.org/10.1007/s10343-004-0044-9.
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Auerbach, Anna B. „Production of functional transgenic mice by DNA pronuclear microinjection.“ Acta Biochimica Polonica 51, Nr. 1 (31.03.2004): 9–31. http://dx.doi.org/10.18388/abp.2004_3593.
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Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integration into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal--in vivo analysis of the function of the gene or gene network. The purpose of this review is to look at the current state of transgenic technology, utilizing a pronuclear microinjection method as the most accepted way of gene transfer into the mouse genome.
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Elson, D. A., und M. S. Bartolomei. „A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.“ Molecular and Cellular Biology 17, Nr. 1 (Januar 1997): 309–17. http://dx.doi.org/10.1128/mcb.17.1.309.
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The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.
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Faerman, A., I. Barash, R. Puzis, M. Nathan, D. R. Hurwitz und M. Shani. „Dramatic heterogeneity of transgene expression in the mammary gland of lactating mice: a model system to study the synthetic activity of mammary epithelial cells.“ Journal of Histochemistry & Cytochemistry 43, Nr. 5 (Mai 1995): 461–70. http://dx.doi.org/10.1177/43.5.7730585.
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We studied the expression of human serum albumin (HSA) driven by the ovine beta-lactoglobulin promoter in the mammary glands of lactating mice from five independent transgenic strains, by employing combined in situ hybridization and immunostaining techniques. Four strains displayed a heterogeneous pattern of expression: mice of strains 91 and 92 expressed the transgene in only a fraction of the lobules, whereas in strains 69 and 83 all lobules contained cells expressing HSA. In all four strains the patterns of expression within expressing lobules corresponded to the morphology of alveolar cells and to the extent of local milk secretion, suggesting that filling of alveolus with secreted material was accompanied by asynchronous downregulation of transgene expression. In situ hybridization to the endogenous milk protein genes alpha-lactalbumin, beta-casein, and whey acidic protein revealed a uniform pattern of expression in lactating mammary glands of transgeneic and in four out of five non-transgeneic mice. In the fifth control mouse, we detected downregulation of gene expression in lobules containing alveoli distended by secreted milk. The pattern of expression of the three endogenous genes was greatly disturbed after a short (3-hr) unilateral closure of mammary glands, and very much resembled the pattern of expression of the HSA transgenes. These results demonstrate that transgeneic mice provide a useful model to study the factors that regulate the synthetic activity of mammary epithelial cells.
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Dunnick, Wesley A., John T. Collins, Jian Shi, Gerwin Westfield, Clinton Fontaine, Paul Hakimpour und F. Nina Papavasiliou. „Switch recombination and somatic hypermutation are controlled by the heavy chain 3′ enhancer region“. Journal of Experimental Medicine 206, Nr. 12 (02.11.2009): 2613–23. http://dx.doi.org/10.1084/jem.20091280.
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Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3′ enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3′ heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3′ enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3′ enhancer region, CSR to most heavy chain genes is reduced to ∼1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3′ enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.
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Palauqui, Jean-Christophe, und Hervé Vaucheret. „Transgenes are dispensable for the RNA degradation step of cosuppression“. Proceedings of the National Academy of Sciences 95, Nr. 16 (04.08.1998): 9675–80. http://dx.doi.org/10.1073/pnas.95.16.9675.
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Cosuppression results in the degradation of RNA from host genes and homologous transgenes after transcription in the nucleus. By using grafting experiments, we have shown previously that a systemic signal mediates the propagation of cosuppression of Nia host genes and 35S-Nia2 transgenes from silenced 35S-Nia2 transgenic stocks to nonsilenced 35S-Nia2 transgenic scions but not to wild-type scions. Here, we examined the requirements for triggering and maintenance of cosuppression in various types of scions. Grafting-induced silencing occurred in 35S-Nia2 transgenic lines over-accumulating Nia mRNA whether they are able to spontaneously trigger cosuppression or not and in 35S-Nia2 transgene-free plants over-accumulating host Nia mRNA caused by metabolic derepression. When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in the 35S-Nia2 transgene-free plants and in the 35S-Nia2 transgenic lines that are not able to trigger cosuppression spontaneously. Conversely, silencing was maintained in the 35S-Nia2 transgenic lines that are able to trigger cosuppression spontaneously. Our results indicate that the presence of a 35S-Nia2 transgene is dispensable for the RNA degradation step of posttranscriptional silencing when host Nia mRNA over-accumulate above the level of wild-type plants. They also suggest that grafting-induced RNA degradation does not result in the production of the systemic silencing signal required for spontaneous triggering and maintenance.
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Lochmüller, E. M., A. Weusten, E. Wolf, E. Cindik, I. Renner-Müller, T. Eikmeier und F. Eckstein. „Geschlechtsspezifische Analyse der Knochenmasse normaler und Wachstumshormon-transgener Mäuse mittels Zweienergie-Röntgen-Absorptiometrie (DXA)“. Annals of Anatomy - Anatomischer Anzeiger 181, Nr. 2 (März 1999): 191–98. http://dx.doi.org/10.1016/s0940-9602(99)80007-7.
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Ray, PC, MS Alam und MS Islam. „Detection of Transgene in Tilapia (Oreochromis niloticus) by Polymerase Chain Reaction (PCR) Technique“. Journal of Environmental Science and Natural Resources 8, Nr. 2 (29.02.2016): 47–49. http://dx.doi.org/10.3329/jesnr.v8i2.26864.
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Polymerase Chain Reaction (PCR) technique using specific primer can be used to detect transgenes. The present study was undertaken to detect salmon growth hormone (GH) gene in transgenic tilapia (Oreochromis niloticus) by PCR. DNA was extracted from F1 Tilapia generated by crossing transgenic parents. Two primers were designed to amplify a part of the region of GH gene sequence, which was used to make transgenic tilapia. To confirm the specificity of the selected primer, PCR was performed on diluted DNAs, extracted from tilapia fin tissues. GH transgene sequences (1500 bp) were successfully amplified from transgenic fish in this study. The specificity of the primers was found to be high in detecting the salmon GH transgenes. The PCR-based method therefore, could be used for fast and easy screening of transgenic fish for this gene.J. Environ. Sci. & Natural Resources, 8(2): 47-49 2015
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Ju Kim, H., K. i. Naruse, W. S. Choi, K. S. Im, C. S. Park und D. I. Jin. „332 ENHANCEMENT OF GROWTH PERFORMANCE IN DOUBLE TRANSGENIC MICE WITH GROWTH HORMONE RECEPTOR AND IGF-1 RECEPTOR GENES“. Reproduction, Fertility and Development 17, Nr. 2 (2005): 317. http://dx.doi.org/10.1071/rdv17n2ab332.
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The effect of amplifying growth-related receptor signaling, through overexpression of receptors, on growth regulation in animals was examined. Transgenic mice lines were produced by DNA microinjection using the metallothionein promoter ligated to either the growth hormone receptor (GHR) or IGF-1 receptor (IGF-1R) genes (3 GHR founders and 3 IGF-1R founders). Transgenic mouse lines were estimated to contain approximately 4 to 20 copies of transgenes per cell by Southern blot analysis. Founder mice of each transgenic line transmitted transgenes into F1 and F2 pups with Mendelian ratio. Double transgenic (IGF-1R/GHR) mice were produced by the mating between nine pairs of IGF-1R and GHR hemizygous transgenic F1 mice. The transmission patterns in the 78 F2 pups produced from these matings were 20 with no transgene (25.6%), 17 with the IGF-1R gene (21.8%), 25 with the GHR gene (32.1%), and 16 with both GHR and IGF-1R genes (20.5%). The mRNA expression of transgenes using RT-PCR with the specific primers for IGF-IR and GHR genes was checked in tissues of transgenic mice. Double transgenic mice with IGF-IR and GHR genes expressed more mRNAs of transgenes than non-transgenic or single transgenic mice. Growth of double transgenic mice was fastest compared with single transgenic mice containing IGF-1R or GHR genes. And GHR transgenic mice grew faster than IGF-1R transgenic mice. When body weights of 15 transgenic mice for each transgenic line were measured at 4, 10, and 14 weeks after birth, double transgenic mice were significantly heavier compared with non-transgenic control mice at each stage (24 to 30% heavier in double transgenic mice; 15 to 20% heavier in single transgenic mice, P < 0.05). These results suggest that overexpression of growth-related receptor genes could promote the growth of transgenic animals with an additive effect.
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Kwan, H., V. Pecenka, A. Tsukamoto, T. G. Parslow, R. Guzman, T. P. Lin, W. J. Muller, F. S. Lee, P. Leder und H. E. Varmus. „Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.“ Molecular and Cellular Biology 12, Nr. 1 (Januar 1992): 147–54. http://dx.doi.org/10.1128/mcb.12.1.147.
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The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.
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Kwan, H., V. Pecenka, A. Tsukamoto, T. G. Parslow, R. Guzman, T. P. Lin, W. J. Muller, F. S. Lee, P. Leder und H. E. Varmus. „Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice“. Molecular and Cellular Biology 12, Nr. 1 (Januar 1992): 147–54. http://dx.doi.org/10.1128/mcb.12.1.147-154.1992.
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The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.
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Carlson, D. F., J. R. Dobrinsky und S. C. Fahrenkrug. „327 HIGH EFFICIENCY SWINE CLONING USING MONOGENIC AND POLYGENIC POOLS OF GENETICALLY MODIFIED CELLS“. Reproduction, Fertility and Development 23, Nr. 1 (2011): 260. http://dx.doi.org/10.1071/rdv23n1ab327.
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Somatic cell nuclear transfer (SCNT) of genetically modified (GM) cells is currently the most widely applied method for the creation of transgenic swine. However, significant clone-to-clone variation in the efficiency of SCNT for a variety of genetically modified cell lines is commonly observed and contributes to the expense of transgenic animal production. A retrospective look at our own results based on the use of monoclonal GM cell lines as donors revealed a dismal efficiency of only 13% (15 embryo transfers resulting in only 2 full term pregnancies). Thus, while SCNT of individual GM clones offers the perceived advantage of prior characterisation of transgene expression or structure, the variability of clonability for any given cell line adds risk to SCNT. In contrast, rates of pregnancy when we used pools of GM cells as donors for SCNT were much better (9 full term pregnancies from 11 transfers, ∼82% efficiency). Four of these litters were derived from polyclonal but monogenic GM cell populations constitutively expressing either human APOBEC3G or YFP-Cre transgenes integrated using the Sleeping Beauty transposon system. Four litters relied on a novel approach, wherein different polyclonal and monogenic GM cell populations (containing different transgenes) were mixed before being used as donors for SCNT. For example, 2 litters were derived from the pooling of 2 GM cell populations carrying different tetracycline inducible or repressible shRNA transgenes, resulting in founders harboring each of the shRNA transgenes (4 in total). Two litters were also created from a pool of 3 distinct polyclonal cell populations, each harboring a different Cre-lox regulated transgene, resulting in the birth of 11 live piglets with founders corresponding to each of the transgenes. Thus, both mono- and polygenic pooling of GM cells significantly enhances the success of SCNT, largely avoiding variation in cell clonability. Furthermore, pooling results in a significant reduction in the time and number of surrogates required to generate a diversity of genetically modified pigs. Importantly, the use of Sleeping Beauty transgene integration resulted in a high rate of transgene-expressing founders. Where expected, the gene of interest transgenes were expressed in 23 of 29 founders (79%), whereas selectable marker transgene expression was observed in 35 of 40 founders (88%). The combination of efficient SCNT from polyclonal and polygenic cell populations and the high proportion of expressers delivered by Sleeping Beauty transgene integration offer a high-efficiency, low-risk solution to swine transgenesis.
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Porter, S. D., und C. J. Meyer. „A distal tyrosinase upstream element stimulates gene expression in neural-crest-derived melanocytes of transgenic mice: position-independent and mosaic expression“. Development 120, Nr. 8 (01.08.1994): 2103–11. http://dx.doi.org/10.1242/dev.120.8.2103.
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We have assessed the importance of a melanocyte-specific DNase I hypersensitive site and matrix attachment region situated 15 kb upstream of the mouse tyrosinase gene by analysis in transgenic mice. Transgenes containing all, part, or none of this region linked to the tyrosinase promoter and human tyrosinase cDNA were introduced into genetically albino mice, and pigmentation and transgene message levels were analyzed in the resulting transgenic lines. The effect of the upstream region was to enhance significantly gene expression in melanocytes, and to provide position-independent expression of the transgene. Two exceptions to complete position independence were seen; these lines displayed a mosaic expression pattern in which the transgene was expressed fully in some melanocyte clones but less so in others, resulting in transverse stripes of colours ranging from near white to dark grey. Unexpectedly, pigmentation in the eye of all transgenic lines containing the upstream region was nonuniform, in that the neural-crest-derived melanocytes of the choroid and anterior iris contained significantly more pigment than those derived from the optic cup (retinal pigment epithelium and posterior iris). Transgenes containing a small part or none of the upstream region were expressed poorly and in a position-dependent manner; of those lines that were visibly pigmented, expression was equal in the neural crest and optic-cup-derived cells of the eye.(ABSTRACT TRUNCATED AT 250 WORDS)
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Seda, Marian, Emma Peskett, Charalambos Demetriou, Dale Bryant, Gudrun E. Moore, Philip Stanier und Dagan Jenkins. „Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages“. F1000Research 8 (11.03.2019): 273. http://dx.doi.org/10.12688/f1000research.17314.1.
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Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.
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Song, Guo-qing, Aaron E. Walworth und Wayne H. Loescher. „Grafting of Genetically Engineered Plants“. Journal of the American Society for Horticultural Science 140, Nr. 3 (Mai 2015): 203–13. http://dx.doi.org/10.21273/jashs.140.3.203.
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Grafting is a well-established agricultural practice, and it now has implications for the commercialization of transgenic plants. In transgrafted plants, only one part (scion or rootstock) is transgenic with the other part untransformed. However, transgenes may affect both mobile and immobile endogenous metabolites (e.g., RNAs, proteins, and phytohormones) and mobility has implications for transgrafting. In the phloem, long-distance transport of mobile metabolites can play important roles in plant development and signaling. In a transgrafted plant, an immobile transgene product (ITP) is not likely to be translocated across the graft union. In contrast, mobile transgene products (MTP) may be translocated across the graft. Regardless of the mobility of transgene products (TP), interaction of transgenic and nontransgenic parts in transgrafted plants through either the MTP or ITP has been demonstrated to be effective in facilitating changes in nontransgenic portions of the plant. Consequently, and particularly in fruit crops, transgrafting provides the potential for improving products from their nontransgenic parts with the possibility of minimizing the controversy over transgenic crops. This review focuses mainly on the mobility of TP and effects on the whole transgrafted plant.
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Smith, Charles. „Integration of transgenes in breeding programs“. BSAP Occasional Publication 12 (1988): 71–80. http://dx.doi.org/10.1017/s0263967x00003293.
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ABSTRACTConventional animal breeding is in a healthy state, with good rates of genetic change possible, and economic efficiency being continually improved in the different livestock species. New reproductive techniques offer faster rates of change, as with the use of embryo transfer in sheep and cattle breeding. It is now possible to introduce foreign DNA, transgenes, into the germ line of farm animals and this offers new opportunities in the genetic improvement of economic merit. The transgenes should be directed at traits of high economic value, and be made in elite breeding stocks to benefit from past genetic improvements. Much still needs to be learned about engineering transgenes and about the basic biochemical-physiological processes they are designed to affect. However many transgenes will be produced over the next decade, and some may be useful in animal improvement. Founder transgene individuals are unique for incorporation site, copy number and expression, and will require screening, multiplication, testing and evaluation, in both hemizygous (TO) and homozygous (TT) form, for all economic traits. For a transgene with a large useful net effect on economic merit, the most rapid genetic response will come from fixing it in the elite breeding stock. An effect of 5-10 percent in economic merit will be needed to offset the normal genetic improvement displaced. Current experience with transgenes is of very large effects on the target trait, but with deleterious effects on fitness and economic merit. Control of the level, tissue and time of expression are crucial to the practical use of transgenes. With uncertainty in the results of a transgenic program, conventional programs will have to be maintained, so extra investment will be required. Patent rights may bring extra benefits to breeders, but there are also risks if the stock prove defective. Breeding strategies and organization may also change. Despite initial problems and uncertainties, the power of the transgenic methods is so great that the development of transgenic stocks is likely to become a very important tool in genetic improvement of livestock in the future.
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Gama, L. T., C. Smith und J. P. Gibson. „Transgene effects, introgression strategies and testing schemes in pigs“. Animal Science 54, Nr. 3 (Juni 1992): 427–40. http://dx.doi.org/10.1017/s0003356100020894.
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AbstractStrategies of introgressing a transgene into a pig nucleus undergoing mass selection for net merit were deterministically evaluated. They consisted of systematically backcrossing hemizygous transgenic sires to females from the selection nucleus, or vice versa, followed by intercrossing of hemizygous individuals, assuming different levels of heritability (h2), polygenic breeding value of the founder animal, inbreeding depression and constraints in availability of resources. The polygenic breeding value of the founder transgenic animal and inbreeding depression were of negligible importance if backcrossing lasted for at least three generations, and there was little advantage in extending backcrossing much further. The best introgression strategy examined was to backcross selection nucleus sires to hemizygous females, but this was a less efficient strategy in terms of testing transgene effects. Testing the survival of homozygous carriers requires approximately five and 100 matings among hemizygous individuals to detect a reduction in viability of 0·5 and 0·1, respectively. Comparing several candidate transgenes in the first generations of backcrossing is feasible, and does not result in substantial delays in improvement of polygenic breeding value in the selected transgene. If resources are limited, the magnitude of the transgene effect (as a proportion of the mean) that compensates for the genetic lag incurred by its introgression is about 0·1 for most economic traits in pigs. To compensate for less selection while backcrossing and for risk in use, transgenes must have an appreciable effect on economic merit to make their introgression worthwhile, even when the additional costs of transgene production are ignored.
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Laible, G., S. A. E. Cole, B. K. Brophy, M. J. Wright, M. C. Berg, A. A. Cullum, S. R. Delaney et al. „335 GENETIC ENGINEERING OF GOATS FOR THE PRODUCTION OF A BIOSIMILAR ANTIBODY IN MILK“. Reproduction, Fertility and Development 25, Nr. 1 (2013): 315. http://dx.doi.org/10.1071/rdv25n1ab335.
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Dairy animals provide an attractive production platform for biosimilar antibodies due to the high protein production capacity of the mammary gland and easy access to milk. Goats are well suited for this approach as they offer a relatively short gestation time and good milk yield and are fully validated for the production of recombinant therapeutics. To generate transgenic goats capable of producing a biosimilar version of cetuximab, a monoclonal antibody for epidermal growth factor receptor and approved for the treatment of specific cancers, we co-transfected primary female fetal fibroblasts with expression constructs for cetuximab’s heavy (HC) and light (LC) chains under the control of the goat β-casein regulatory sequences. Beta-globin insulators were added to both transgenes to minimize position effects, and an antibiotic selection marker was placed downstream of the HC transgene sequences to allow for the isolation of stable transgenic cell clones. Selected cell clones were screened by PCR for the presence of both transgenes. Positive cell clones were analysed by Southern blot with a β-casein-specific probe. This allowed for the simultaneous detection of both transgenes, and the endogenous β-casein gene served as a standard to determine transgene copy numbers. The cell clones showed a broad range of copy numbers, from single copy insertions to >100 copies for the HC and LC transgenes. Interestingly, most of the cell clones had more LC than HC transgene copies. Ten cell clones were selected to generate transgenic founders using somatic cell nuclear transfer. We were able to produce 43 live kids from 9 cell lines following transfer of between 26 and 153 one- and two-cell embryos per line into recipients (range of 4 to 15 embryos per recipient). The one cell clone that we used unsuccessfully had the lowest number of transferred embryos (11). The efficiency for the production of live kids per transferred embryos was, on average, 5.1% (range of 1.0 to 9.7%). Kids from 5 lines were hormonally induced into lactation at the age of 10 weeks. Two lines with high copy numbers (≥30) produced either no or only a few drops of milk, whereas the lines with ≤25 transgene copies gave up to several milliliters of milk per day. Western analyses confirmed cetuximab production levels of 15 g L–1 in 2 of the lines with ≤25 transgene copies and ~45 g L–1 in a high copy number line; one low copy number line showed good HC but very low LC expression. Our data demonstrate that cetuximab can be produced in significant quantities in transgenic goats. Future work is aimed at determining production levels under natural lactation conditions and characterising glycosylation patterns to fully understand the pharmacodynamic properties of the antibody. Supported by GTC, the NZ Ministry of Science and Innovation and AgResearch.
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Starck, J., R. Sarkar, M. Romana, A. Bhargava, AL Scarpa, M. Tanaka, JW Chamberlain, SM Weissman und BG Forget. „Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region“. Blood 84, Nr. 5 (01.09.1994): 1656–65. http://dx.doi.org/10.1182/blood.v84.5.1656.1656.
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Abstract Two lines of transgenic mice carrying a normal 40-kb Kpn I beta-globin cluster transgene lacking the locus control region (LCR) were analyzed for the expression of human gamma- and beta-globin genes during mouse development. After RNase protection assays, the ratios of human G gamma- , A gamma-, or beta-mRNAs relative to endogenous mouse zeta + alpha mRNAs were obtained for each stage of development. The two gamma transgenes were expressed in day-11.5 blood (embryonic stage) and day- 13.5 blood (early fetal stage), but their expression was markedly decreased by day 16.5 of fetal life. Expression of the beta transgene was essentially absent at day 13.5, appeared at a low level by day 16.5, and was maximal by day 18.5, reaching a level similar to that observed in adult mice. Therefore, developmentally regulated expression of the human gamma- and beta-globin transgenes was obtained in the absence of the LCR. The relative expression of human gamma- and beta- globin genes was also examined in mice carrying 40-kb Kpn I beta- cluster transgenes with two different base substitutions associated with nondeletion forms of hereditary persistence of fetal hemoglobin (HPFH), -202 C-->G G gamma HPFH and -117 G-->A A gamma HPFH. The ratio of G gamma- to beta-globin transcripts was markedly increased in red blood cells of adult mice from three different lines carrying the transgene with the -202 G gamma HPFH mutation. This result confirms our previous preliminary results (Tanaka et al: Ann NY Acad Sci, 612:167, 1990) indicating that the -202 G gamma HPFH phenotype was reproduced in transgenic mice. The relatively low levels of G gamma-mRNA expression in adult mice carrying the non-HPFH transgene excludes a major influence of the 3′ beta-globin enhancer, present upstream of the G gamma gene because of the tandem repeat insertion, as a factor in the persistent G gamma gene expression observed in blood of adult mice carrying the -202 G gamma HPFH transgene. This conclusion is also supported by the fact that, in mice carrying the -117 A gamma HPFH transgene, G gamma-globin mRNA was detected in blood of adult animals only at low levels similar to that observed in the non-HPFH lines. However, the A gamma-HPFH phenotype was not reproduced in the transgenic lines carrying the -117A gamma HPFH mice.
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Starck, J., R. Sarkar, M. Romana, A. Bhargava, AL Scarpa, M. Tanaka, JW Chamberlain, SM Weissman und BG Forget. „Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region“. Blood 84, Nr. 5 (01.09.1994): 1656–65. http://dx.doi.org/10.1182/blood.v84.5.1656.bloodjournal8451656.
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Two lines of transgenic mice carrying a normal 40-kb Kpn I beta-globin cluster transgene lacking the locus control region (LCR) were analyzed for the expression of human gamma- and beta-globin genes during mouse development. After RNase protection assays, the ratios of human G gamma- , A gamma-, or beta-mRNAs relative to endogenous mouse zeta + alpha mRNAs were obtained for each stage of development. The two gamma transgenes were expressed in day-11.5 blood (embryonic stage) and day- 13.5 blood (early fetal stage), but their expression was markedly decreased by day 16.5 of fetal life. Expression of the beta transgene was essentially absent at day 13.5, appeared at a low level by day 16.5, and was maximal by day 18.5, reaching a level similar to that observed in adult mice. Therefore, developmentally regulated expression of the human gamma- and beta-globin transgenes was obtained in the absence of the LCR. The relative expression of human gamma- and beta- globin genes was also examined in mice carrying 40-kb Kpn I beta- cluster transgenes with two different base substitutions associated with nondeletion forms of hereditary persistence of fetal hemoglobin (HPFH), -202 C-->G G gamma HPFH and -117 G-->A A gamma HPFH. The ratio of G gamma- to beta-globin transcripts was markedly increased in red blood cells of adult mice from three different lines carrying the transgene with the -202 G gamma HPFH mutation. This result confirms our previous preliminary results (Tanaka et al: Ann NY Acad Sci, 612:167, 1990) indicating that the -202 G gamma HPFH phenotype was reproduced in transgenic mice. The relatively low levels of G gamma-mRNA expression in adult mice carrying the non-HPFH transgene excludes a major influence of the 3′ beta-globin enhancer, present upstream of the G gamma gene because of the tandem repeat insertion, as a factor in the persistent G gamma gene expression observed in blood of adult mice carrying the -202 G gamma HPFH transgene. This conclusion is also supported by the fact that, in mice carrying the -117 A gamma HPFH transgene, G gamma-globin mRNA was detected in blood of adult animals only at low levels similar to that observed in the non-HPFH lines. However, the A gamma-HPFH phenotype was not reproduced in the transgenic lines carrying the -117A gamma HPFH mice.
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Simmons, Michael J., Kevin J. Haley, Craig D. Grimes, John D. Raymond und Joseph C. L. Fong. „Regulation of P-Element Transposase Activity in Drosophila melanogaster by hobo Transgenes That Contain KP Elements“. Genetics 161, Nr. 1 (01.05.2002): 205–15. http://dx.doi.org/10.1093/genetics/161.1.205.
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Abstract Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry+, Δ2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry+, Δ2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA.
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Moeller, Marcus J., Abdulsalam Soofi, Silja Sanden, Jürgen Floege, Wilhelm Kriz und Lawrence B. Holzman. „An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo“. American Journal of Physiology-Renal Physiology 289, Nr. 2 (August 2005): F481—F488. http://dx.doi.org/10.1152/ajprenal.00332.2004.
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The utility of promoter fragments isolated from the 5′-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (β-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas β-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (β-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39–42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.
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Kong, Siyuan, Jinxue Ruan, Kaiyi Zhang, Bingjun Hu, Yuzhu Cheng, Yubo Zhang, Shulin Yang und Kui Li. „Kill two birds with one stone: making multi-transgenic pre-diabetes mouse models through insulin resistance and pancreatic apoptosis pathogenesis“. PeerJ 6 (17.04.2018): e4542. http://dx.doi.org/10.7717/peerj.4542.
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Background Type 2 diabetes is characterized by insulin resistance accompanied by defective insulin secretion. Transgenic mouse models play an important role in medical research. However, single transgenic mouse models may not mimic the complex phenotypes of most cases of type 2 diabetes. Methods Focusing on genes related to pancreatic islet damage, peripheral insulin resistance and related environmental inducing factors, we generated single-transgenic (C/EBP homology protein, CHOP) mice (CHOP mice), dual-transgenic (human islet amyloid polypeptide, hIAPP; CHOP) mice (hIAPP-CHOP mice) and triple-transgenic (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1; hIAPP; CHOP) mice (11β-HSD1-hIAPP- CHOP mice). The latter two types of transgenic (Tg) animals were induced with high-fat high-sucrose diets (HFHSD). We analyzed the diabetes-related symptoms and histology features of the transgenic animals. Results Comparing symptoms on the spot-checked points, we determined that the triple-transgene mice were more suitable for systematic study. The results of intraperitoneal glucose tolerance tests (IPGTT) of triple-transgene animals began to change 60 days after induction (p < 0.001). After 190 days of induction, the body weights (p < 0.01) and plasma glucose of the animals in Tg were higher than those of the animals in Negative Control (Nc). After sacrificed, large amounts of lipid were found deposited in adipose (p < 0.01) and ectopically deposited in the non-adipose tissues (p < 0.05 or 0.01) of the animals in the Tg HFHSD group. The weights of kidneys and hearts of Tg animals were significantly increased (p < 0.01). Serum C peptide (C-P) was decreased due to Tg effects, and insulin levels were increased due to the effects of the HFHSD in the Tg HFHSD group, indicating that damaged insulin secretion and insulin resistance hyperinsulinemia existed simultaneously in these animals. The serum corticosterone of Tg was slightly higher than those of Nc due to the effects of the 11βHSD-1 transgene and obesity. In Tg HFHSD, hepatic adipose deposition was more severe and the pancreatic islet area was enlarged under compensation, accompanying apoptosis. In the transgenic control diet (Tg ControlD) group, hepatic adipose deposition was also severe, pancreatic islets were damaged, and their areas were decreased (p < 0.05), and apoptosis of pancreatic cells occurred. Taken together, these data show the transgenes led to early-stage pathological changes characteristic of type 2 diabetes in the triple-transgene HFHSD group. The disease of triple-transgenic mice was more severe than that of dual or single-transgenic mice. Conclusion The use of multi-transgenes involved in insulin resistance and pancreatic apoptosis is a better way to generate polygene-related early-stage diabetes models.
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Oke, Krista B., Peter A. H. Westley, Darek T. R. Moreau und Ian A. Fleming. „Hybridization between genetically modified Atlantic salmon and wild brown trout reveals novel ecological interactions“. Proceedings of the Royal Society B: Biological Sciences 280, Nr. 1763 (22.07.2013): 20131047. http://dx.doi.org/10.1098/rspb.2013.1047.
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Interspecific hybridization is a route for transgenes from genetically modified (GM) animals to invade wild populations, yet the ecological effects and potential risks that may emerge from such hybridization are unknown. Through experimental crosses, we demonstrate transmission of a growth hormone transgene via hybridization between a candidate for commercial aquaculture production, GM Atlantic salmon ( Salmo salar ) and closely related wild brown trout ( Salmo trutta ). Transgenic hybrids were viable and grew more rapidly than transgenic salmon and other non-transgenic crosses in hatchery-like conditions. In stream mesocosms designed to more closely emulate natural conditions, transgenic hybrids appeared to express competitive dominance and suppressed the growth of transgenic and non-transgenic (wild-type) salmon by 82 and 54 per cent, respectively. To the best of our knowledge, this is the first demonstration of environmental impacts of hybridization between a GM animal and a closely related species. These results provide empirical evidence of the first steps towards introgression of foreign transgenes into the genomes of new species and contribute to the growing evidence that transgenic animals have complex and context-specific interactions with wild populations. We suggest that interspecific hybridization be explicitly considered when assessing the environmental consequences should transgenic animals escape to nature.
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Pinheiro, Patrícia Valle, Josias Corrêa de Faria, Elsa Oliveira Paranaguá e. Lago Nogueira und Francisco José Lima Aragão. „Transgene inheritances and genetic similarities of near isogenic lines of genetically modified common beans“. Pesquisa Agropecuária Brasileira 44, Nr. 9 (September 2009): 1168–76. http://dx.doi.org/10.1590/s0100-204x2009000900015.
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The objective of the present work was to determine the inheritance and stability of transgenes of a transgenic bean line expressing the genes rep-trap-ren from Bean golden mosaic virus and the bar gene. Crosses were done between the transgenic line and four commercial bean cultivars, followed by four backcrosses to the commercial cultivars. Progenies from each cross were evaluated for the presence of the transgenes by brushing the leaves with glufosinate ammonium and by polymerase chain reaction using specific oligonucleotides. Advanced generations were rub-inoculated with an isolate of Bean common mosaic necrosis virus (BCMNV). The transgenes were inherited consistently in a Mendelian pattern in the four crosses studied. The analyzed lines recovered close to 80% of the characteristics of the recurrent parent, as determined by the random amplified DNA markers used, besides maintaining important traits such as resistance to BCMNV. The presence of the transgene did not cause any detectable undesirable effect in the evaluated progenies.
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Cuthbert, Janice L., Peter B. E. McVetty, Georges Freyssinet und Martine Freyssinet. „Comparison of the performance of bromoxynil-resistant and susceptible near-isogenic populations of oilseed rape“. Canadian Journal of Plant Science 81, Nr. 3 (01.07.2001): 367–72. http://dx.doi.org/10.4141/p00-115.
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Bromoxynil herbicide resistance is the newest type of broad-spectrum, non-selective herbicide resistance to be introduced into oilseed rape (Brassica napus L.). This herbicide resistance is conferred by a single transgene (the oxy gene), taken from a soil bacterium, which confers the ability to metabolize hydroxybenzonitrile herbicides such as bromoxynil. The level of resistance to bromoxynil herbicide in oilseed rape is high, but it is not known whether there are any performance changes associated with the oxy transgene or with the derived herbicide resistance. To determine if there are changes in performance related to the oxy transgene, or the derived herbicide resistance, two near-isogenic transgenic bromoxynil-resistant populations, with two different forms of the oxy gene, Westar 235 and Westar 237, were developed at the University of Manitoba, grown in the field in Manitoba for several years and evaluated for comparative performance. Westar 235 and Westar 237 near-isogenic populations, either sprayed with bromoxynil at 280 g a.i. ha–1 or left unsprayed, were compared with the non-transgenic near-isogenic population cultivar, Westar, in Winnipeg, Carman, and Portage la Prairie from 1994 to 1997. There were no consistent differences in the performance of the sprayed or unsprayed transgenic near-isogenic populations in comparison to Westar for any trait. The few significant and minimal differences that were found were evenly divided between positive effects and negative effects of the oxy gene and derived herbicide resistance. There were no significant effects of spraying bromoxynil herbicide detected in paired comparisons of Westar 235 and Westar 237 sprayed versus Westar 235 and Westar 237 unsprayed, respectively. The effects of the oxy transgenes and the effects of bromoxynil spraying of herbicide-resistant lines were negligible, indicating that there is little, if any biological cost associated with the bromoxynil resistance transgenes or related herbicide resistance. Key words: Transgenic oilseed rape, Brassica napus, biological cost
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Doglio, Lynn, Joo Yeun Kim, Grazyna Bozek und Ursula Storb. „Expression ofλand K Genes Can Occur in all B Cells and is Initiated Around the Same Pre-B-Cell Developmental Stage“. Developmental Immunology 4, Nr. 1 (1994): 13–26. http://dx.doi.org/10.1155/1994/87352.
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Transgenic mice that carry a λ2 transgene under the control of the Vλ2 promoter and the Eλ2-4 enhancer (λ2Eλ mice) are described. A high proportion of B cells in the spleen and the bone marrow express the λ transgene on the cell membrane. λ2 protein is synthesized by all λ2Eλ-derived spleen B-cell hybridomas that have retained the transgene, suggesting that all B cells have the ability to express λ genes. Feedback inhibition of endogenous K-gene rearrangement is significant, but not complete. The results are similar to those with transgenic mice expressing the same λ2 transgene under the control of the heavy-chain enhancer (λ2EH mice). Although the λ2EH transgene is expressed before the λ2Eλ transgene, feedback inhibition seems to occur at about the same stage of B-cell development, regardless of the timing of expression of the λ transgenes. Apparently, feedback is not necessarily coincident with the assembly of a heavy-chain/light-chain complex in pre-B cells. Expression of λ in the normal fetal liver coincides with the expression of K; thus, it appears that λ-gene transcription is not delayed. The differential rearrangement of K and λ genes is discussed in the light of these findings.
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Martin, Anne F., Sunita Bhatti, Gail J. Pyne-Geithman, Mariam Farjah, Vlasios Manaves, Lori Walker, Roberta Franks, Arthur R. Strauch und Richard J. Paul. „Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle“. American Journal of Physiology-Cell Physiology 293, Nr. 1 (Juli 2007): C238—C245. http://dx.doi.org/10.1152/ajpcell.00567.2006.
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Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH2-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle α-actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 ± 14%, 92 ± 11%) in SM1 and decreased to 57 ± 1% and 80 ± 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 ± 0.3 s) and SM2 slower (7.1 ± 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues.
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Stevenson, Zachary C., Megan J. Moerdyk-Schauwecker, Brennen Jamison und Patrick C. Phillips. „Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans“. G3: Genes|Genomes|Genetics 10, Nr. 10 (19.08.2020): 3775–82. http://dx.doi.org/10.1534/g3.120.401400.
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Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native microhomology-based recombination, to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.
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Byrd, Daniel M., und M. Luann Roegner. „Endocrine Modulation and the Federal Government“. International Journal of Toxicology 17, Nr. 2 (Februar 1998): 111–28. http://dx.doi.org/10.1080/109158198226657.
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Recent events have drawn attention to the hypothesis that some xenobiotics in the environment may elicit toxicities in humans by modulating endocrine pathways. From the perspective of regulatory toxicology, pursuit of this hypothesis w ill prove difficult, because current risk assessment m ethods do not readily apply to substances with very high potencies, reversibility, transgener-ational effects, and subtle biological outcomes. Such xenobiotics typically persist in the body and bioaccumulate in the food chain. Yet the exposures are important only during critical periods of vulnerability, and no sustained bio-markers of these important exposures currently exist. This article describes some recent efforts by federal agencies to pursue the hypothesis, including research planning and screening of potential endocrine modulating xenobiotics and risk assessment of dioxin conflicts with its policy for substances that cause thyroid follicular carcinomas.
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Alimuddin, G. Yoshizaki, O. Carman und T. Takeuchi. „Effectiveness of hCMV, mEF1a and mAct promoters on driving of foreign gene expression in transgenic zebrafish“. Jurnal Akuakultur Indonesia 6, Nr. 1 (01.01.2007): 65. http://dx.doi.org/10.19027/jai.6.65-77.
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<p>Highly unsaturated fatty acids (HUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have long been recognized for its beneficial effect for human health and development. The D6 fatty acid desaturase is generally considered to be the rate-limiting factor in HUFA biosynthesis. Here, as the first step of study, we conducted experiment to select an appropriate construct that allows higher expression levels of masu salmon (<em>Oncorhynchus masou</em>)<em> </em>D6-desaturase gene in zebrafish (<em>Danio rerio</em>) in order to increase its activity for synthesizing EPA/DHA. Salmon D6-desaturase cDNA (sD6) was separately ligated with human cytomegalovirus (hCMV), medaka elongation factor 1a (mEF1a) and medaka b-actin (mAct) promoters. The resulted construct was designated as hCMV-sD6, mEF1a-sD6 and mAct-sD6, respectively. Each of the constructs in circular DNA form was microinjected into 1-cell stage embryos at a concentration of 30mg/ml. Transgenic individuals were identified by polymerase chain reaction (PCR) and their expression levels were analyzed by reverse transcription PCR. The first (F1) and second (F2) generation was produced by crossing the transgenic founder F0 and F1, respectively, with wild-type fish. The results showed that the highest transient gene expression level was obtained from the mAct-D6 construct, followed respectively by EF1a-D6 and hCMV-D6 construct. The transmission rate of transgene into F1 generation was 4.2%-44.1%, and into F2 was followed the Mendellian segregation pattern. Expression of transgene in F2 generation was varied between strains regarding as the mosaics of F0 fish. Now, a transgenic system to study the modification of fatty acid biosynthesis pathways in fish was established. Further investigations are to produce fish containing higher levels of EPA and DHA.</p> <p>Keywords: desaturase, nutraceutical fatty acid, transgenic, zebrafish, masu salmon</p> <p> </p> <p>Abstrak</p> <p>Promoter merupakan regulator yang menentukan tempat, waktu dan tingkat ekspresi gen. Pada penelitian ini, kami melakukan seleksi kontruksi plasmid yang tepat yang menghasilkan tingkat ekspresi yang tinggi dari gen D6-desaturase-like ikan masu salmon (<em>Oncorhynchus masou</em>)<em> </em>yang ditransfer ke ikan zebra (<em>Danio rerio</em>) untuk meningkatkan kemampuannya dalam mensintesa EPA/DHA. cDNA D6-desaturase-like (OmD6FAD) dari ikan salmon masu diligasi secara terpisah dengan promoter dari cytomegalovirus manusia (hCMV), elongation factor 1a (mEF1a) dan b-actin (mbAct) dari ikan medaka, untuk membuat konstruksi plasmid yang berturut-turut disebut sebagai hCMV-OmD6FAD, mEF1a- OmD6FAD dan mbAct-OmD6FAD. Konstruksi tersebut dengan konsentrasi 30mg/ml disuntikkan ke embrio pada saat fase satu sel. Individu transgenik diidentifikasi menggunakan PCR dan tingkat ekspresi transgen dianalisa dengan RT-PCR. Hasil menunjukkan bahwa tingkat ekspresi sementara yang tertinggi dari gen asing adalah diperoleh dari konstruksi mbAct-OmD6FAD, diikuti selanjutnya oleh EF1a-OmD6FAD dan hCMV- OmD6FAD. Transgen telah ditransmisikan ke ikan generasi F2 dengan mengikuti pola segregasi Mendel. Tingkat ekspresi transgen yang tinggi pada jaringan ikan F2 yang diperiksa telah diperoleh. Dengan demikian, sebuah sistem transgenik untuk memodifikasi biosistesa asam lemak pada ikan telah dikembangkan. </p> <p>Kata kunci: promoter, desaturase asam lemak, transgenik, ikan zebra, ikan salmon masu</p>
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Nussenzweig, M. C., A. C. Shaw, E. Sinn, J. Campos-Torres und P. Leder. „Allelic exclusion in transgenic mice carrying mutant human IgM genes.“ Journal of Experimental Medicine 167, Nr. 6 (01.06.1988): 1969–74. http://dx.doi.org/10.1084/jem.167.6.1969.
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Expression of the membrane-bound version of the human mu chain in transgenic mice results in the allelic exclusion of endogenous mouse Ig heavy chain genes (6). The secreted version of the human Ig transgene has no such effect. F1 hybrid animals that carry transgenes for both secreted and membrane-bound human mu chains produce both forms of the human heavy chain while strongly suppressing endogenous mouse mu expression. The simultaneous expression of the two rearranged transgenes in primary B cells suggests that allelic exclusion operates before the formation of a second functionally rearranged heavy chain gene in vivo.
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DeLoia, Julie A., und Davor Solter. „A transgene insertional mutation at an imprinted locus in the mouse genome“. Development 108, Supplement (01.04.1990): 73–79. http://dx.doi.org/10.1242/dev.108.supplement.73.
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Genetic imprinting in mice results in functional differences in the oocyte and spermatocyte genomes, as evidenced by both genetic and pronuclear transfer experiments. To gain insights into the molecular mechansims involved in the imprinting process, researchers have studied methylation phenotypes and expression of hemizygous transgenes associated with parental origin. In this report, we describe a transgenic mouse lineage in which expression of both the transgene and an endogenous gene at the insertion site are determined by the parent of origin. The mutation caused by transgene insertion shows variable expressivity and incomplete penetrance in addition to a modified dominant pattern of inheritance.
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Donoviel, D. B., M. A. Shield, J. N. Buskin, H. S. Haugen, C. H. Clegg und S. D. Hauschka. „Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.“ Molecular and Cellular Biology 16, Nr. 4 (April 1996): 1649–58. http://dx.doi.org/10.1128/mcb.16.4.1649.
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Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.
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Dunnick, Wesley A., Jian Shi, Kevin A. Graves und John T. Collins. „The 3′ end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four γ genes“. Journal of Experimental Medicine 201, Nr. 9 (25.04.2005): 1459–66. http://dx.doi.org/10.1084/jem.20041988.
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The switch in immunoglobulin (Ig) heavy chain class is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and class switch recombination we used transgenic mice with a 230-kilobase bacterial artificial chromosome that included a rearranged VDJ gene and the entire heavy chain constant region locus. In addition to several lines with intact transgenes, we identified two lines in which the heavy chain locus transgene lacked Cα and everything 3′ of it, including the regulatory elements HS3a, HS1-2, HS3b, and HS4. B cells from both lines with the truncated transgenes make abundant transgenic (Tg) VDJCμ transcripts and IgM protein. Deletion of the 3′ end of the locus results in dramatically reduced expression of both germline transcripts and switched VDJCH transcripts of the γ3, γ2b, γ2a, and ε genes. In addition, the transgenes lacking the 3′ end of the locus express reduced amounts of γ1 germline transcripts and 2–3% of the amount of Tg IgG1 in tissue culture compared with intact transgenes. Finally, switch recombination to γ1 is undetectable in the transgenes lacking the 3′ elements, as measured by digestion circularization–polymerase chain reaction or by the expression of VDJCγ1 transcripts.
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Alimuddin, G. Yoshizaki und O. Carman. „Rapid method for identification of transgenic fish zygosity“. Jurnal Akuakultur Indonesia 6, Nr. 2 (01.07.2007): 177. http://dx.doi.org/10.19027/jai.6.177-182.
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<p>Identification of zygosity in transgenik fish is normally achieved by PCR analysis with genomic DNA template extracted from the tissue of progenies which are derived by mating the transgenic fish and wild-type counterpart. This method needs relatively large amounts of fish material and is time- and labor-intensive. New approaches addressing this problem could be of great help for fish biotechnologists. In this experiment, we applied a quantitative real-time PCR (qr-PCR) method to analyze zygosity in a stable line of transgenic zebrafish (<em>Danio rerio</em>) carrying masu salmon, <em>Oncorhynchus masou</em> D6-desaturase-like gene. The qr-PCR was performed using iQ SYBR Green Supermix in the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, USA). Data were analyzed using the comparative cycle threshold method. The results demonstrated a clear-cut identification of all transgenic fish (<em>n</em>=20) classified as a homozygous or heterozygous. Mating of those fish with wild-type had revealed transgene transmission to the offspring following expected Mendelian laws. Thus, we found that the qTR-PCR to be effective for a rapid and precise determination of zygosity in transgenic fish. This technique could be useful in the establishment of breeding programs for mass transgenic fish production and in experiments in which zygosity effect could have a functional impact.</p> <p>Keywords: quantitative real-time PCR; zygosity; transgenic fish; mass production</p> <p> </p> <p>ABSTRAK</p> <p>Identifikasi sigositas ikan transgenik biasanya dilakukan menggunakan analisa PCR dengan cetakan DNA genomik yang diekstraksi dari jaringan ikan hasil persilangan antara ikan transgenik dan ikan normal. Metode ini memerlukan ikan dalam jumlah yang banyak, dan juga waktu serta tenaga. Pendekatan baru untuk mengatasi masalah tersebut akan memberikan manfaat besar kepada peneliti bioteknologi perikanan. Pada penelitian ini, kami menggunakan metode PCR real-time kuantitatif (krt-PCR) untuk menganalisa sigositas pada satu strain ikan zebra (<em>Danio rerio</em>)<em> </em>transgenik yang membawa gen D6-desaturase-like dari ikan salmon masu, <em>Oncorhynchus masou</em>.<em> </em>krt-PCR dilakukan menggunakan <em>iQ SYBR Green Supermix</em> pada mesin <em>iCycler iQ Real-time PCR Detection system</em> (Bio-Rad Laboratories, USA). Data dianalisis menggunakan metode pembandingan nilai <em>cycle threshold</em>. Hasil penelitian menunjukkan bahwa semua ikan transgenik (<em>n</em>=20) yang diidentifikasi dapat diklasifikasikan secara jelas sebagai ikan homosigot atau heterosigot. Persilangan antara ikan transgenik tersebut dengan ikan normal menunjukkan transmisi transgen ke keturunannya mengikuti hukum segregasi Mendel. Dengan demikian, metode krt-PCR adalah efektif untuk penentuan sigositas secara cepat dan tepat pada ikan transgenik. Teknik ini dapat berguna dalam program produksi ikan transgenik secara massal dan dalam percobaan dimana faktor sigositas memberikan pengaruh nyata.</p> Kata kunci: kuantitatif real-time PCR; sigositas, ikan transgenik; produksi massal