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Plant J. 35(2), 273-283. Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. 2003

Yamamoto, Y.Y., Tsuhara, Y., Gohda, K., Suzuki, K. and Matsui, M.

Notes: These researchers used luciferase-containing T-DNA insertions in Arabidopsis thaliana for gene trapping. Luciferase was chosen because its transient expression allowed temporal expression studies. Several insertion vectors were constructed and found to have different insertion frequencies. Vectors containing the luc+ gene had substantially higher insertion rates than native luciferase vectors. Luciferase activity was measured in vivo with a CCD camera or, for longer term studies, with an automated scintillation counter sampling every 15-25 minutes over one week. The application of IRES sites in gene trapping experiments was also investigated using firefly and Renilla luciferases. The Dual-Luciferase® Reporter Assay System was used to monitor luciferase activity in vitro. Finally, to sequence the T-DNA insertion sites, genomic DNA was isolated from T2 seedlings using the Wizard® Magnetic 96 DNA Plant System and was subsequently amplified and sequenced. (2787)

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J. Biol. Chem. 277, 17349-17358. Structural Determinants of BRCA1 Translational Regulation 2002

Sobczak, K. and Krzyzosiak, W.J.

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5' UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5' UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM vector and ligated to PCR amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc).Ten additional cDNAs were made containing a variety of changes to the 5'UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5'UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5'UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

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J. Immunol. 162, 254-262. Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts. 1999

Klavinskis, L.S., Barnfield, C., Gao, L., Parker, S.

Notes: The authors cloned luciferase cDNA from pGEM®-luc Vector into eukaryotic expression vector, then transfected into nasal epithelium of BALB/c mice. They measured luciferase activity using the Luciferase Assay System on tissue extracts. They also used pGEM®-luc Vector to generate fluorescein-labeled antisense luciferase RNA probes for in situ staining. (0906)

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pGEM®-luc DNA

J. Biol. Chem. 273, 14002-14007. 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. 1998

Poulin, F., Gingras, A.C., Olsen, H., Chevalier, S., Sonenberg, N.

Notes: The authors created a bicistronic vector with a CMV-driven expression of Renilla luciferase followed by the IRES sequence from the 5'-UTR of poliovirus type II Lansing and trailed by the wildtype firefly luciferase gene. The construct was cotransfected into HeLa cells with CMV-driven expression vectors containing wildtype eIF-4E binding protein or mutants. The wildtype eIF-4E binding protein reduced Renilla luciferase activity by about 75% but resulted in a two-fold increase in firefly luciferase. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System and the pGEM®-luc Vector was the source of the Firefly luciferase and the pRL-CMV Vector was the source of Renilla luciferase. (0544)

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Gene Ther. 5, 65-75. Inhibition of HIV-1 replication by combined expression of gag dominant negative mutant and a human ribonuclease in a tightly controlled HIV-1 inducible vector 1998

Cara, A., Rybak, S.M., Newton, D.L., Rottschafer, S.E., Reitz Jr., M.S., and Gusella, G.L.

Notes: The ProFection® Mammalian Transfection System - Calcium Phosphate was used to transfect HeLa and HeLa-Tat cell lines. The authors developed a vector-antiviral gene system for use in gene therapy in the treatment of HIV-1. pGEM®-luc was used as a negative control in luciferase assays of transfected cells. (1388)

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J. Biol. Chem. 273, 21663-21668. The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or initiator regions. 1998

Singh, U., Rogers, J.B.

Notes: The RNAgents® Total RNA Isolation System was used to isolate RNA from a nuclear run-on assay from the nuclei harvested from 5 x 107 trophozoites stably transfected with reporter plasmids. The luciferase reporter was constructed using the luciferase cDNA from the pGEM®-luc Vector. (0371)

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pGEM®-luc DNA

J. Biol. Chem. 272, 5936-5942. Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells. 1997

Bai, G. and Kusiak, J.W.

Notes: Reporter vectors (produced with the pGL2 Basic Vector) were transiently transfected into PC12 cells and promoter activity was measured in response to 2.5S NGF. Promoter constructs were generated by PCR, subcloned into the pGEM®-T Vector and sequenced. The pGEM®-luc Vector was used to generated an antisense luc RNA probe used in RNase protection assays (using RNase ONE™ Ribonuclease) to follow the transcription of luciferase DNA following NGF treatment. The early growth reaction transcription factor and variants were expressed in the TNT® SP6 Coupled Reticulocyte Lysate System and used to perform gel shifts with promoter elements. The SP1 Consensus Oligonucleotide was used to compete transcription factor binding to the promoter elements. (1493)

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J. Cell Biol. 138, 1343-1354. Tissue-specific expression of the L1 cell adhesion molecule is modulated by the neural restrictive silencer element. 1997

Kallunki, P., Edelman, G.M. and Jones, F.S.

Notes: Studies were performed in NIH 3T3 and Neuro2a cells. The pGEM®-luc Vector was used as a source of the luciferase gene to convert previous beta-Galactosidase reporter vector constructs to luciferase reporter vectors. The TnT® Coupled Reticulocyte Lysate System was used to verify that the cloned NRSF/REST protein could be expressed intact. (1566)

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Proc. Natl. Acad. Sci. USA 94, 8812-8817. Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica 1997

Singh, U., Rogers, J. B., Mann, B. J. , Petri, W. A., Jr

Notes: The RNAgents® Total RNA Isolation System and the PolyATtract® mRNA Isolation System were used to isolate total and polyA+ RNA, respectively, from Entamoeba histolytica. The total RNA was used for nuclear run-on transcription. The polyA+ RNA was used for Northerns and primer extensions. The pGEM®-luc Vector was used to produce an amoeboid reporter vector. (0370)

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Proc. Natl. Acad. Sci. USA 94(16), 8812-8817. Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica. 1997

Singh, U., Rogers, J.B., Mann, B.J. and Petri, W.A., Jr.

Notes: Poly A+ RNA was isolated directly from E. histolytica using the PolyATtract® System 1000 and used for Northern and primer extension analysis. The RNAgents® Total RNA Isolation System was used to isolate total RNA from E. histolytica. The isolated RNA was used for nuclear run-on transcription. (1654)

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