Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter inLactobacillus casei

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.

Download Full-text

Green Fluorescent Protein (GFP) Expression in Mammalian Cells After UV-Irradiation

Fundamentals for the Assessment of Risks from Environmental Radiation ◽

10.1007/978-94-011-4585-5_40 ◽

1999 ◽

pp. 311-316

Author(s):

M. Palm ◽

C. Baumstark-Khan ◽

G. Horneck

Keyword(s):

Green Fluorescent Protein ◽

Uv Irradiation ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Gfp Expression ◽

Green Fluorescent ◽

Expression In Mammalian Cells

Download Full-text

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000445950 ◽

2016 ◽

Vol 26 (4) ◽

pp. 284-290 ◽

Cited By ~ 1

Author(s):

Lin Liu ◽

Xiangyan Zhang ◽

Siyuan Yang ◽

Yao Zhai ◽

Weijia Liu ◽

...

Keyword(s):

Flow Cytometry ◽

Green Fluorescent Protein ◽

Promoter Activity ◽

Expression Vector ◽

Fluorescent Protein ◽

Gfp Expression ◽

Qrt Pcr ◽

Key Factor ◽

Green Fluorescent

Aims: The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. Methods: The blaCTX-M promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of blaCTX-M in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. Results: The promoters of all the blaCTX-M genes were provided by ISEcp1 and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of blaCTX-M was influenced. Conclusion: The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the blaCTX-M gene does not specifically enhance the expression of blaCTX-M, contrary to previous reports. The expression of blaCTX-M may be regulated by changes in promoter activity caused by diverse spacer sequences.

Download Full-text

Visualizing Viral Dissemination in the Mouse Nervous System, Using a Green Fluorescent Protein-Expressing Borna Disease Virus Vector

Journal of Virology ◽

10.1128/jvi.00098-10 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5438-5442 ◽

Cited By ~ 14

Author(s):

Andreas Ackermann ◽

Timo Guelzow ◽

Peter Staeheli ◽

Urs Schneider ◽

Bernd Heimrich

Keyword(s):

Nervous System ◽

Green Fluorescent Protein ◽

Disease Virus ◽

Fluorescent Protein ◽

Gfp Expression ◽

Borna Disease Virus ◽

Viral Dissemination ◽

Viral Spread ◽

Green Fluorescent ◽

Borna Disease

ABSTRACT Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.

Download Full-text

Transcriptional analyses of an ethanol inducible promoter inEscherichia coliand tobacco for production of cellulase and green fluorescent protein

Biotechnology & Biotechnological Equipment ◽

10.1080/13102818.2015.1065711 ◽

2015 ◽

Vol 29 (6) ◽

pp. 1043-1052 ◽

Cited By ~ 1

Author(s):

Homa Hemmati ◽

Chhandak Basu

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Inducible Promoter ◽

Green Fluorescent

Download Full-text

Transformation of Escherichia coli K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiology†

Journal of Food Protection ◽

10.4315/0362-028x-69.2.276 ◽

2006 ◽

Vol 69 (2) ◽

pp. 276-281 ◽

Cited By ~ 11

Author(s):

T. P. OSCAR ◽

K. DULAL ◽

D. BOUCAUD

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Predictive Models ◽

Fluorescent Protein ◽

Inducible Promoter ◽

E Coli ◽

Contaminated Food ◽

Green Fluorescent ◽

K 12 ◽

High Copy Plasmid

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (μmax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40°C, μmax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

Download Full-text

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.

Download Full-text

Efficient genetic transformation of Sorghum using a visual screening marker

Genome ◽

10.1139/g04-095 ◽

2005 ◽

Vol 48 (2) ◽

pp. 321-333 ◽

Cited By ~ 47

Author(s):

Zhensheng Gao ◽

J Jayaraj ◽

S Muthukrishnan ◽

Larry Claflin ◽

G H Liang

Keyword(s):

Green Fluorescent Protein ◽

Transgenic Plants ◽

Reporter Gene ◽

Target Gene ◽

Fluorescent Protein ◽

Efficient Production ◽

Gfp Expression ◽

Visual Screening ◽

Green Fluorescent ◽

Screening Marker

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.Key words: Agrobacterium tumefaciens, green fluorescent protein (GFP), sorghum transformation.

Download Full-text