The Human Eukaryotic Initiation Factor 4AI Gene (EIF4A1) Contains Multiple Regulatory Elements That Direct High-Level Reporter Gene Expression in Mammalian Cell Lines

Quinn, Carmel M.; Wiles, Alan P.; El‐Shanawany, Tariq; Catchpole, I.; Alnadaf, Tanya; Ford, M. J.; Gordon, Siamon; Greaves, David R.

doi:10.1006/geno.1999.6031

Cited by 16 publications

(17 citation statements)

References 18 publications

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“…All enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The eIF4A1 promoter (0.5 kb) from BamHI/ HindIII-digested pEIF4A1-blunt 60 (provided by Jonathan Hardy and Michael Bachmann, Stanford University) was cloned into BglII/HindIII-digested pCMV-hMGFP/CBL resulting in pTD153. The Ubc promoter was prepared by digestion of pTEJ-8 61 (provided by Thomas Kledal and Michael Bachmann) with NheI followed by Klenow treatment to generate 'blunt ends.'…”

Section: Plasmidsmentioning

confidence: 99%

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

González-González

Spitler

et al. 2010

Gene Ther

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Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

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Section: Plasmidsmentioning

confidence: 99%

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

González-González

Spitler

et al. 2010

Gene Ther

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“…Human MMP-2 Promoter Constructs-Luciferase reporter plasmids were constructed by cloning 3 concatenated copies of the adjacent 24 nucleotides, flanking a variant, upstream of a previously characterized minimal promoter region of the human translation initiation factor EIF-4AI gene (30). KpnI sites were introduced at the 5Ј-and 3Ј-ends of concatamers to facilitate cloning.…”

Section: Isolation Of the Human Mmp-2 Promoter-the Human Genomementioning

confidence: 99%

“…Transfection was carried out using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals) according to the manufacturer's protocol. Cells were cotransfected with 0.5 g of reporter plasmid and 0.1 g of pcDNA3-␤-galactosidase expression vector (30) to standardize for transfection efficiency. Cells were incubated for 24 h, washed twice in phosphate-buffered saline, and harvested by the addition of 100 l of lysis buffer (Luciferase Assay System, Promega).…”

Section: Isolation Of the Human Mmp-2 Promoter-the Human Genomementioning

confidence: 99%

Identification of Novel, Functional Genetic Variants in the Human Matrix Metalloproteinase-2 Gene

Price¹,

Greaves

Watkins³

2001

Journal of Biological Chemistry

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375

313

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Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. We scanned a 2-kilobase pair promoter region and all 13 exons of the human MMP-2 gene, from a panel of 32 individuals, and we identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5-untranslated region, 6 in the coding region, 1 in intronic sequence, and 1 in the 3-untranslated region. The majority of coding region polymorphisms resulted in synonymous substitutions, whereas three promoter variants (at ؊1306, ؊790, and ؉220) mapped onto cis-acting elements. We functionally characterized all promoter variants by transient transfection experiments with 293, RAW264.7, and A10 cells. The common C 3 T transition at ؊1306 (allele frequency 0.26), which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. Electrophoretic mobility shift assays confirmed that these differences in allelic expression were attributable to abolition of Sp1 binding. These data suggest that this common functional genetic variant influences MMP-2 gene transcription in an allele-specific manner and is therefore an important candidate to test for association in a wide spectrum of pathologies for which a role for MMP-2 is implicated, including atherogenesis and tumor invasion and metastasis.

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“…Notably, the EF-1a promoter induced higher levels of transgene expression than the viral CMV promoter in (Byun et al, 2005). The efficiency of cellular promoters has been shown to be cell specific and dependent on cell activity (Doll et al, 1996;Durieux et al, 2005;Ramezani et al, 2000;Spenger et al, 2004). While studies indicated that osteoblastic specific promoters, including the promoter of the osteocalcin gene, led to increase in transgene expression over time for BMP-2 expression in MSCs (Kumar et al, 2005), the effect of various cellular and tissue-specific promoters on the level and duration of BMP-2 expression in MSCs has not yet been well investigated.…”

Section: Introductionmentioning

confidence: 99%

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

Ferreira

Potier

Vaudin

et al. 2012

eCM

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Document VersionPublisher's PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication:• A submitted manuscript is the author's version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication Citation for published version (APA):Ferreira, E., Potier, E., Vaudin, P., Oudina, K., Bensidhoum, M., Logeart-Avramoglou, D., ... Petite, H. (2012). Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after bmp-2 transgene electrotransfer. European Cells and Materials, 24, 18-28. General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. AbstractTransplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66-and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficie...

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The Human Eukaryotic Initiation Factor 4AI Gene (EIF4A1) Contains Multiple Regulatory Elements That Direct High-Level Reporter Gene Expression in Mammalian Cell Lines

Cited by 16 publications

References 18 publications

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

Identification of Novel, Functional Genetic Variants in the Human Matrix Metalloproteinase-2 Gene

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

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