scholarly journals Effectiveness of B-actin promoter on driving target gene expression in common carp transgenesis

2011 ◽  
Vol 10 (1) ◽  
pp. 16
Author(s):  
Andi Aliah Hidayani ◽  
Odang Carman ◽  
. Alimuddin
Keyword(s):  
Gene Expression ◽  
Common Carp ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Transgenic Fish ◽  
Hatching Rate ◽  
Gfp Expression ◽  
Cell Stage ◽  
Target Gene Expression ◽  
Actin Promoter

<p>Promoter in transgene construct plays an important role on regulating of transgene expression level in transgenic fish. In fish transgenesis, researcher convinced that use all-fish gene construct is safety and prospective. This study was performed to compare effectiveness b-actin promoter, - the promoter which has ubiquitous, constitutive, housekeeping characteristics, from common carp (homologous) and from tilapia and medaka b-actin promoters (heterologous) in driving of green fluorescent protein (GFP) expression as a model of target gene on common carp<em> </em>transgenesis. These gene constructs were separately microinjected into cytoplasm of 60 one-cell-stage common carp embryos. The results suggested that 70% survival rate at embryo stage and 45% hatching rate values showed that the microinjection was performed successfully. Percentage of embryos expressing GFP gene were slightly higher when injected using common carp and medaka promoters than those of using tilapia promoter. Percentage of larvae expressing GFP using common carp promoter was similar with medaka promoter. Furthermore, GFP expression using common carp b-actin promoter could be detected at one-week-old larvae, while GFP expressing using medaka b-actin promoter was lasted at 2-day-old larvae. The results demonstrated that homologous promoter more effective in driving of a target gene expression than that of heterologous promoter. </p> <p>Key words: homologous promoter, GFP, transgenesis, common carp</p> <p> </p> <p>ABSTRAK</p> <p>Promoter dalam konstruksi transgen berperan penting dalam pengaturan tingkat ekspresi transgen pada ikan transgenik. Dalam transgenesis ikan, peneliti meyakini bahwa penggunaan konstruksi gen "all-fish" adalah aman dan prospektif.  Penelitian ini dilakukan untuk membandingkan efektivitas promoter β-aktin, - promoter yang memiliki ciri <em>ubiquitous</em>, <em>constitutive</em>, dan <em>housekeeping</em>, dari ikan dari ikan mas (homolog) dan ikan nila dan ikan medaka (heterolog) dalam mengendalikan ekspresi gen GFP sebagai model gen pada transgenesis ikan mas. Setiap  konstruksi gen tersebut diinjeksikan secara terpisah ke sitoplasma embrio ikan mas fase 1 sel sebanyak 60 embrio. Hasil penelitian dengan kelangsungan hidup embrio 70% dan derajat penetasan 45% menunjukkan bahwa kegiatan mikroinjeksi berhasil dengan baik.  Persentase embrio mengekspresikan gen GFP yang diinjeksi konstruksi gen dengan promoter β-aktin ikan mas dan ikan medaka sedikit lebih tinggi dibandingkan dengan yang menggunakan promoter β-aktin ikan nila.  Selanjutnya, ekspresi gen GFP yang dikendalikan oleh promoter β-aktin ikan mas dapat dideteksi pada larva berumur 1 minggu, sedangkan ekspresi GFP dengan promoter β-aktin ikan medaka hanya bisa terdeteksi hingga larva berumur 2 hari.  Hasil penelitian menunjukkan bahwa promoter homolog adalah lebih efektif dalam mengatur ekspresi gen target dibandingkan dengan promoter heterolog.</p> <p>Kata kunci: promoter homolog, GFP, transgenesis, ikan mas</p>

21 SITE-SPECIFIC RECOMBINATION USING Dre-RECOMBINASE IN PORCINE CELLS AND EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
S. Y. Yum ◽  
S. J. Kim ◽  
J. H. Moon ◽  
W. J. Choi ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cellular Level ◽  
Cell Stage ◽  
Site Specific ◽  
Target Gene Expression ◽  
Green Fluorescent ◽  
Gateway Cloning System

Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.

63 ESTABLISHMENT OF GREEN FLUORESCENT PROTEIN EXPRESSED DOG CELL LINES CONTROLLED BY DOXYCYCLINE

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
S. G. Hong ◽  
J. E. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Cell Size ◽  
Fluorescent Protein ◽  
Biomedical Science ◽  
Gfp Expression ◽  
Cell Stage ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

An inducible gene expression system in transgenic animals has been widely used in biomedical science. The aim of this study was to establish green fluorescent protein (GFP) inducible dog cell line and evaluate the system in embryos using interspecies somatic cell nuclear transfer (iSCNT). Canine fetal fibroblasts were transfected with retroviral vector containing GFP, rtTA, and TRE and designated Gteton cell line. For iSCNT, bovine ovaries were collected from a local slaughterhouse and COCs were matured for 24 h. The denuded oocytes were enucleated, injected with Gteton cells, treated with 24 h of doxycycline (DOX), and electrically fused (NEPA GENE, 34 V, 15 μs, 2 pulses). The reconstructed oocytes were activated and then cultured in modified SOF medium. To verify the stability of the Gteton cells, 2 experiments were designed. Experiment 1 was designed to compare the cell size and viability of Gteton and nontransfected cells. Countness™ (Invitrogen, version 1.0, Carlsbad, CA, USA) was used for analysis. In experiment 2, the control of GFP gene expression was observed when the cells were cultured with 1 mg mL-1 of DOX. The cells were also cultured without DOX after 24 h of DOX treatment. Photographs were taken of cultured cells every 12 h. The intensity of GFP expression was analyzed by using Image J freeware (U.S. National Institutes of Health, version 1.42, NIH, Bethesda, MD, USA). To evaluate the reprogramming ability of the Gteton cells in embryos, another 2 experimental designs were planned. Experiment 3 estimated GFP expression in iSCNT embryos when they were cultured with and without DOX. Experiment 4 assessed the development of the iSCNT embryos under microscopy. Data were analyzed using statistical analysis system program (version 9.1, SAS Institute, Cary, NC, USA). In experiment 1, there was no significance (P < 0.05) in average viable cell size (13.7 v. 13.2 μm) or viability (97.0 v. 98.7%). In experiment 2, the GFP intensity increased steadily when cultured in medium containing DOX. The intensity was increased approximately two times after 24 h compared with 12 h of treatment. The intensity after 24 h of DOX treatment decreased to the basal level after 5 days. In experiment 3, the GFP intensity of iSCNT embryos cultured in mSOF containing DOX was increased approximately two times in 16-cell stage compared with 2-cell stage. In experiment 4, the cleavage rate was not significantly different between the 2 groups. In conclusion, we dtermined that the inducible system of Gteton cell line was established in a stable manner. Furthermore the results from iSCNT may indicate the possibility to produce GFP-expressed transgenic puppies controlled by doxycyline. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

scholarly journals Aktivitas Promoter â-aktin Ikan Medaka Jepang (Oryzias latipes) pada Ikan Mas (Cyprinus carpio)

2012 ◽  
Vol 11 (2) ◽  
pp. 70
Author(s):  
Alimuddin ' ◽  
Lola Irma Purwanti ◽  
MH. Fariduddin Ath-thar ◽  
Chairul Muluk ◽  
Odang Carman ◽  
...  
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Fluorescent Protein ◽  
Oryzias Latipes ◽  
Green Fluorescent Protein Gene ◽  
Hatching Rate ◽  
Cell Stage ◽  
Larval Stages ◽  
Renilla Reniformis ◽  
Green Fluorescent

This study was conducted to examine activity of medaka (Oryzias latipes) â-actin promoter (mBP) in common carp(Cyprinus carpio) as the first step towards development of common carp transgenic in country. Gene constructpmBP-hrGFP that consists of mBA promoter and humanized Renilla reniformis green fluorescent protein gene(hrGFP) was injected into cytoplasm of one cell stage of common carp by using microinjector. PmBP-hrGFPconcentration used for microinjection was 50 μg/mL aquabides. Parameters observed were survival rate of embryo(SRe), hatching rate (HR) and expression of hrGFP gene. SRe was calculated before eggs hacthed, while hatchingrate (HR) was after all of eggs hatched. The activity of mBA promoter was analyzed by observation of hrGFP genetransient expression using a fluorescence microscope. The results of experiment showed that SRe (87,5%) andHR (79.2%) of control was respectevily higher than that of injected treatment (75.0% & 61.7%). Expression of hrGFPwas observed firstly at blastula (12 hours after fertilization) to 1-day-old larval stages (24 hours after hatching)with higher gene expression at blastula to late gastrula stages. Percentage of micronjected larvae expressinghrGFP at 6 hours after hatching reached 71.6 ± 6.7%. Conclusion was that mBA promoter could drove hrGFPexpression in common carp, hence it can be used to produce common carp transgenic by changing hrGFP withgenes correlated with important traits in aquaculture.

2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 2049-P
Author(s):  
REBECCA K. DAVIDSON ◽  
NOLAN CASEY ◽  
JASON SPAETH
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Nucleosome Remodeling ◽  
Target Gene Expression

scholarly journals On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

2021 ◽  
Author(s):  
Philipp Moritz Fricke ◽  
Angelika Klemm ◽  
Michael Bott ◽  
Tino Polen
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Literature Search ◽  
Target Gene ◽  
Target Genes ◽  
Recombinant Dna ◽  
Acetic Acid Bacteria ◽  
Homoserine Lactone ◽  
Expression Systems ◽  
Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

2002 ◽  
Vol 88 (2) ◽  
pp. 363-371 ◽  
Author(s):  
Aruna V. Krishnan ◽  
Donna M. Peehl ◽  
David Feldman
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Vitamin D ◽  
Target Gene ◽  
Cancer Growth ◽  
Target Gene Expression

Regulation of barley miRNAs upon dehydration stress correlated with target gene expression

2010 ◽  
Vol 10 (4) ◽  
pp. 493-507 ◽  
Author(s):  
Melda Kantar ◽  
Turgay Unver ◽  
Hikmet Budak
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Dehydration Stress ◽  
Target Gene Expression

Regulation of Wnt Signaling Target Gene Expression by the Histone Methyltransferase DOT1L

2014 ◽  
Vol 10 (1) ◽  
pp. 109-114 ◽  
Author(s):  
Garrett S. Gibbons ◽  
Scott R. Owens ◽  
Eric R. Fearon ◽  
Zaneta Nikolovska-Coleska
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Target Gene ◽  
Histone Methyltransferase ◽  
Target Gene Expression
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