Firefly luciferase gene: structure and expression in mammalian cells.

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.

Download Full-text

Facilitated DNA transfer to rat submandibular gland in vivo and GRP-Ca gene regulation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1995.268.6.g1074 ◽

1995 ◽

Vol 268 (6) ◽

pp. G1074-G1078 ◽

Cited By ~ 1

Author(s):

B. C. O'Connell ◽

K. G. Ten Hagen ◽

K. W. Lazowski ◽

L. A. Tabak ◽

B. J. Baum

Keyword(s):

Luciferase Activity ◽

Firefly Luciferase ◽

Upstream Region ◽

Luciferase Expression ◽

Upstream Sequence ◽

Luciferase Gene ◽

Base Pairs ◽

Firefly Luciferase Gene ◽

Rat Submandibular Gland

The internalization of DNA can be facilitated by adenovirus infection. Using the replication-deficient adenovirus, Ad-dl312, and a plasmid-based firefly luciferase gene as a reporter, we have optimized the uptake and expression of DNA in rat submandibular glands in vivo. Luciferase expression is transient and peaked at approximately 18 h after infection. Luciferase activity increased with plasmid concentration and was greatest at 10(9) to 10(10) plaque-forming units of Ad-dl312 per gland. We next examined the expression in vivo of plasmids containing deletions of the glutamine/glutamic acid-rich protein (GRP-Ca isoform) gene upstream region linked to a chloramphenicol acetyltransferase (CAT) reporter. Constructs with 9.4, 6.3, and 2.7 kb and 17 base pairs of upstream sequence gave relative CAT activities of 100, 30, 7.6, and 38.5, respectively. With the 9.4-kb GRP-Ca construct, CAT was preferentially expressed in acinar cells, which is characteristic of GRP. This gene transfer approach should prove useful in the further study of gene expression in salivary glands and other organs.

Download Full-text

Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

Biochemical Journal ◽

10.1042/bj2760637 ◽

1991 ◽

Vol 276 (3) ◽

pp. 637-641 ◽

Cited By ~ 51

Author(s):

F F Craig ◽

A C Simmonds ◽

D Watmore ◽

F McCapra ◽

M R H White

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Firefly Luciferase ◽

Luciferase Expression ◽

Intact Cells ◽

Cos Cells ◽

Escherichia Coli Cells ◽

Luminescent Signal ◽

The Difference ◽

Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Download Full-text

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

Journal of General Virology ◽

10.1099/0022-1317-83-6-1311 ◽

2002 ◽

Vol 83 (6) ◽

pp. 1311-1314 ◽

Cited By ~ 11

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.

Download Full-text

The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

Journal of General Virology ◽

10.1099/vir.0.83293-0 ◽

2008 ◽

Vol 89 (1) ◽

pp. 336-340 ◽

Cited By ~ 21

Author(s):

Esther Schnettler ◽

Hans Hemmes ◽

Rob Goldbach ◽

Marcel Prins

Keyword(s):

Mammalian Cells ◽

Firefly Luciferase ◽

Hek293 Cells ◽

Rnai Pathway ◽

Luciferase Expression ◽

Hairpin Rna ◽

Rnai Suppressor ◽

Ns3 Protein ◽

Rnai Response

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed.

Download Full-text