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

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.

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Green fluorescent protein (GFP) as a direct biosensor for mutation detection: Elimination of false-negative errors in target gene expression

Analytical Biochemistry ◽

10.1016/j.ab.2008.05.029 ◽

2008 ◽

Vol 380 (1) ◽

pp. 91-98 ◽

Cited By ~ 6

Author(s):

Yu Kyung Tak ◽

Pravin K. Naoghare ◽

Kyeong-Hee Lee ◽

Soon-Sup Park ◽

Joon Myong Song

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Target Gene ◽

Mutation Detection ◽

Fluorescent Protein ◽

False Negative ◽

Target Gene Expression ◽

Green Fluorescent

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

Jurnal Akuakultur Indonesia ◽

10.19027/jai.10.16-23 ◽

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

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 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. Key words: homologous promoter, GFP, transgenesis, common carp ABSTRAK 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 ubiquitous, constitutive, dan housekeeping, 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. Kata kunci: promoter homolog, GFP, transgenesis, ikan mas

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Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic stripe expression in the developing Drosophila epidermis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.16.8606 ◽

1997 ◽

Vol 94 (16) ◽

pp. 8606-8611 ◽

Cited By ~ 34

Author(s):

G. Vorbruggen ◽

H. Jackle

Keyword(s):

Gene Expression ◽

Target Gene ◽

Attachment Site ◽

Muscle Attachment ◽

Site Specific ◽

Specific Target ◽

Target Gene Expression ◽

Specific Target Gene

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab63 ◽

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.

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Translational Control using an Expanded Genetic Code

International Journal of Molecular Sciences ◽

10.3390/ijms20040887 ◽

2019 ◽

Vol 20 (4) ◽

pp. 887 ◽

Cited By ~ 1

Author(s):

Yusuke Kato

Keyword(s):

Gene Expression ◽

Genetic Code ◽

Translational Control ◽

Target Gene ◽

Unnatural Amino Acid ◽

High Yield ◽

Site Specific ◽

Stop Codons ◽

Target Gene Expression ◽

Wide Range

A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation.

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

Diabetes ◽

10.2337/db20-2049-p ◽

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

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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11269-z ◽

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.

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