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

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

Immunology Letters ◽

10.1016/j.imlet.2017.12.005 ◽

2018 ◽

Vol 194 ◽

pp. 29-39 ◽

Cited By ~ 10

Author(s):

Fatemeh Motevalli ◽

Azam Bolhassani ◽

Shilan Hesami ◽

Sepideh Shahbazi

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Green Fluorescent

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A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants

Biochemical Journal ◽

10.1042/bj20021966 ◽

2003 ◽

Vol 373 (2) ◽

pp. 403-408 ◽

Cited By ~ 67

Author(s):

Nadya G. GURSKAYA ◽

Arkady F. FRADKOV ◽

Natalia I. POUNKOVA ◽

Dmitry B. STAROVEROV ◽

Maria E. BULINA ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Random Mutagenesis ◽

Wild Type ◽

Green Luminescence ◽

Mammalian Cell Lines ◽

Biological Tool ◽

Fluorescent Pattern ◽

Green Fluorescent

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

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

Omni-Akuatika ◽

10.20884/1.oa.2018.14.2.556 ◽

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.

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High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells

Blood ◽

10.1182/blood.v90.9.3316 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3316-3321 ◽

Cited By ~ 53

Author(s):

Ana Limón ◽

Javier Briones ◽

Teresa Puig ◽

Mercé Carmona ◽

Oscar Fornas ◽

...

Keyword(s):

Flow Cytometry ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Hematopoietic Cells ◽

Retroviral Vectors ◽

Green Fluorescent Protein Gene ◽

Efficient System ◽

Green Fluorescent

Abstract Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34+ cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

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