Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter

Virus Genes ◽  
1992 ◽  
Vol 6 (4) ◽  
pp. 379-386 ◽  
Author(s):  
Tamara N. Kopylova-Sviridova ◽  
Valentina I. Krauzova ◽  
Tatyana M. Timiryasova ◽  
Tatyana V. Gorelova ◽  
Nikolai G. Shuppe ◽  
...  
Keyword(s):  
Transient Expression ◽  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Transient Expression Assay ◽  
Firefly Luciferase Gene ◽  
Baculovirus System

scholarly journals Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes

2002 ◽  
Vol 83 (6) ◽  
pp. 1311-1314 ◽  
Author(s):  
Hendrik J. Rademaker ◽  
Mohamed A. Abou El Hassan ◽  
Gijs A. Versteeg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Hepg2 Cells ◽  
Adenovirus Type ◽  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Wild Type ◽  
Genome Packaging ◽  
Firefly Luciferase Gene ◽  
Adenovirus Vectors ◽  
Initiation Of Dna Replication ◽  
Adenovirus Type 5

Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a ΔE1ΔE3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing ΔE1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.

scholarly journals Firefly luciferase gene: structure and expression in mammalian cells.

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

scholarly journals Firefly luciferase gene contains a cryptic promoter

RNA ◽  
2008 ◽  
Vol 14 (9) ◽  
pp. 1720-1729 ◽  
Author(s):  
V. Vopalensky ◽  
T. Masek ◽  
O. Horvath ◽  
B. Vicenova ◽  
M. Mokrejs ◽  
...  
Keyword(s):  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Cryptic Promoter ◽  
Firefly Luciferase Gene

Firefly luciferase gene: structure and expression in mammalian cells

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Cytokinin-Enhanced Regeneration of Plants From Microprojectile Bombarded Sugarcane Meristematic Tissue

1994 ◽  
Vol 21 (5) ◽  
pp. 603 ◽  
Author(s):  
RL Gambley ◽  
JD Bryant ◽  
NP Masel ◽  
GR Smith
Keyword(s):  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Target Tissue ◽  
Luciferase Gene ◽  
Meristematic Tissue ◽  
Sugarcane Cultivar ◽  
Shoot Initiation ◽  
Cytokinin Treatment ◽  
Suitable Target ◽  
Firefly Luciferase Gene

Sugarcane plants expressing the firefly luciferase gene were regenerated from microprojectile-bombarded meristematic tissue. The concentration and type of cytokinin added to the basal media, both prior to bombardment and during regeneration, significantly affected the number of plantlets produced and the percentage of plantlets expressing luciferase. Maximum shoot initiation from meristematic tissue of sugarcane cultivar Q137 was achieved on media supplemented with N6-benzylaminopurine (BAP). There were differences in the numbers of shoots initiated from different sugarcane cultivars, and some optimisation for each cultivar was required. No luciferase activity was observed in plantlets regenerated in the absence of cytokinin. Sugarcane meristematic tissue preconditioned by exposure to cytokinin appears to be a suitable target tissue for the introduction of genes of agronomic importance into sugarcane. This approach should be readily adaptable to most commercial varieties of sugarcane with minor modification to the cytokinin treatment necessary for optimal regeneration.

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