scholarly journals Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

2009 ◽  
Vol 28 (2) ◽  
pp. 149-155 ◽  
Author(s):  
Kelly S. Santangelo ◽  
Sarah A. Baker ◽  
Gerard Nuovo ◽  
Jonathan Dyce ◽  
Jeffrey S. Bartlett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Guinea Pig ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Full Thickness ◽  
Adeno Associated Virus ◽  
Virus Serotype

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

Changes in the wheat Em 5′ UTR affect reporter gene expression in vivo and in vitro

2001 ◽  
Vol 158 (7) ◽  
pp. 929-934 ◽  
Author(s):  
Manjiri R. Paradkar ◽  
William R. Marcotte
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression

Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo

1999 ◽  
Vol 72 (1-2) ◽  
pp. 125-133 ◽  
Author(s):  
C Watkins ◽  
S Lau ◽  
R Thistlethwaite ◽  
J Hopkins ◽  
G.D Harkiss
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Afferent Lymph

scholarly journals Selective modulation of the SM22α promoter by the binding of BTEB3 (basal transcription element-binding protein 3) to TGGG repeats

2003 ◽  
Vol 375 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Karen M. MARTIN ◽  
Peter D. ELLIS ◽  
James C. METCALFE ◽  
Paul R. KEMP
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Reporter Gene ◽  
Binding Protein ◽  
Reporter Gene Expression ◽  
Promoter Activation ◽  
Basal Transcription ◽  
Element Binding Protein

We have previously identified a C2H2 zinc-finger transcription factor [BTEB3 (basal transcription element-binding protein 3)/KLF13 (Krüppel-like factor 13)] that activates the minimal promoter for the smooth muscle-specific SM22α gene in other types of cell. We show that recombinant BTEB3 binds to three TGGG motifs in the minimal SM22α promoter. By mutation analysis, only one of these boxes is required for BTEB3-dependent promoter activation in P19 cells and BTEB3 activates or inhibits reporter gene expression depending on the TGGG box to which it binds. Transient transfection experiments show that BTEB3 also activates reporter gene expression from the SM22α promoter in VSMCs (vascular smooth muscle cells). Similar studies showed that BTEB3 did not activate expression from the promoter regions of the smooth muscle myosin heavy chain or smooth muscle α-actin promoters, which contain similar sequences, implying that promoter activation by BTEB3 is selective. The expression of BTEB3 is readily detectable in VSMCs in vitro and is modulated in response to injury in vivo.

scholarly journals Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure

Gut ◽  
1999 ◽  
Vol 44 (6) ◽  
pp. 800-807 ◽  
Author(s):  
S Wirtz ◽  
P R Galle ◽  
M F Neurath
Keyword(s):  
Gene Expression ◽  
Gene Delivery ◽  
Lamina Propria ◽  
Reporter Gene ◽  
Mononuclear Cells ◽  
Reporter Gene Expression ◽  
Local Administration ◽  
Fibre Structure

BACKGROUND/AIMSReplication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.METHODSAn E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. β-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.RESULTSIntravenous or intraperitoneal injection of AdCMVβGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVβGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVβGal caused a higher β-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMVβGal vectors.CONCLUSIONSLocal administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.

scholarly journals Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Gwang Sik Kim ◽  
Young Chul Lee
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Reporter Gene Expression ◽  
Temperature Sensitive ◽  
Internal Deletion ◽  
Cell Extracts ◽  
Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

scholarly journals In Vivo Analysis of Glucocorticoid-Induced Reporter Gene Expression Using Gene Gun DNA Delivery

2002 ◽  
Vol 25 (8) ◽  
pp. 1115-1118 ◽  
Author(s):  
Kiyoshi Tanigawa ◽  
Katsunao Tanaka ◽  
Hidetaka Nagase ◽  
Hidekazu Miyake ◽  
Mamoru Kiniwa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Dna Delivery ◽  
Reporter Gene Expression ◽  
Gene Gun ◽  
In Vivo Analysis

scholarly journals The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

2006 ◽  
Vol 80 (19) ◽  
pp. 9831-9836 ◽  
Author(s):  
Bassel Akache ◽  
Dirk Grimm ◽  
Kusum Pandey ◽  
Stephen R. Yant ◽  
Hui Xu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cultured Cells ◽  
Human Gene ◽  
Laminin Receptor ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Tumor Gene

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

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