300 DEVELOPMENT OF TETRACYCLINE-REGULATED GREEN FLUORESCENT PROTEIN EXPRESSING TRANSGENIC RATS BY LENTIVIRAL VECTOR

2009 ◽  
Vol 21 (1) ◽  
pp. 247
Author(s):  
C. Agca ◽  
A. Chan ◽  
Y. Agca
Keyword(s):  
Green Fluorescent Protein ◽  
Blot Analysis ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Sprague Dawley ◽  
Transgenic Rats ◽  
Gfp Gene ◽  
Green Fluorescent ◽  
Kidney Liver

Although various methodologies have been available for genetic modification in mouse, such genomic tools are very limited in rats. The aim of the present study was to take advantage of lentiviral gene delivery to develop effective inducible germline transgenic rats. A tetracycline inducible system was designed to control the expression of the green fluorescent protein (GFP) gene. The GFP gene was constructed downstream of tetracycline response element under the control of a mini cytomegalovirus promoter. Additionally, rtTA2s-Ms was constructed at the 3 prime terminus of GFP under the control of an ubiquitin promoter. The resulted construct was named pLV-Tet-GFP. The pLV-Tet-GFP lentiviral vectors were injected into perivitelline space of 115 Sprague-Dawley rat zygotes. Of those 115 zygotes injected, 93 zygotes were transferred into 4 surrogate mothers and 32 live pups were obtained. The presence of transgene in Fo generation was first detected by PCR and subsequently Southern blot analysis in 25 out of 32 pups (78%). A varying level of GFP expression was observed in the 14 Fo pups after feeding them with 2 g L–1 of tetracycline via drinking water for 4 days. Northern blot analysis was performed in the subsequent generations to determine spatial (brain, kidney, liver, and lung tissues) and temporal (day 0, day 4, day 11, and day 30) expression. The results showed expression intensity as being highest in kidney, brain, lung, and liver. There was little expression detected on day 11 and 30. This study suggests that lentiviral vectors can be used to deliver inducible system quite efficiently in rats. The authors thank Dr. Carlos Lois for providing the lentiviral backbone, Dr. W. Hillen for providing the rtTA2s-Ms fragment, and GuoFu Fang for developing the inducible lentiviral vector.

scholarly journals Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Liyue Wang ◽  
Kao Zhang ◽  
Hongyu Lin ◽  
Wenyan Li ◽  
Jiexia Wen ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
North American ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Type Ii ◽  
Nsp2 Gene ◽  
Gfp Gene ◽  
Overlap Pcr ◽  
Green Fluorescent ◽  
North American Type

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

scholarly journals Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Omni-Akuatika ◽  
2018 ◽  
Vol 14 (2) ◽  
Author(s):  
Eni Kusrini ◽  
Alimuddin Alimuddin ◽  
Erma Primanita Hayuningtyas ◽  
Syuhada Restu Danupratama
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Betta Splendens ◽  
Foreign Gene ◽  
Gfp Expression ◽  
Dna Concentration ◽  
Gfp Gene ◽  
Green Fluorescent

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species.  This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model.  Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment.  Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent.  Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%).  Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds.  A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation.  The successful of foreign gene transfer was determined by PCR method with GFP specific primers.  The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%.  Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%.  The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%.  Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods. 

scholarly journals Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 406-413 ◽  
Author(s):  
Yoichi Ueta ◽  
Hiroaki Fujihara ◽  
Ryota Serino ◽  
Govindan Dayanithi ◽  
Hitoshi Ozawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Physiological Role ◽  
Transgenic Rats ◽  
Patch Clamp Technique ◽  
Axon Terminals ◽  
Green Fluorescent ◽  
Vasopressin Neurons

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

scholarly journals A vector carrying the GFP gene (Green fluorescent protein) as a yeast marker for fermentation processes

2000 ◽  
Vol 57 (4) ◽  
pp. 713-716 ◽  
Author(s):  
Luiz Humberto Gomes ◽  
Keila Maria Roncato Duarte ◽  
Felipe Gabriel Andrino ◽  
Ana Maria Brancalion Giacomelli ◽  
Flavio Cesar Almeida Tavares
Keyword(s):  
Green Fluorescent Protein ◽  
Early Detection ◽  
Fluorescent Protein ◽  
Low Frequency ◽  
Yeast Cells ◽  
Gfp Gene ◽  
Spoilage Yeasts ◽  
Adh2 Promoter ◽  
Pure Cultures ◽  
Green Fluorescent

Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3). Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.

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