Quantitative bioluminescence imaging of transgene expression in vivo

Rettig, Garrett R.; McAnuff, Marie A.; Liu, Dijie; Kim, Jinhyun; Rice, Kevin G.

doi:10.1016/j.ab.2006.04.026

Cited by 48 publications

(52 citation statements)

References 17 publications

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“…[52] BLI is rapid, sensitive and does not generate false positives. [47,52] In addition, Rettig et al have shown that luciferase expression measured by tissue harvesting and luciferase expression measured using BLI are directly correlated. This therefore eliminates the need to harvest tissue and allows for the same reduced number of animals to be assayed over time.…”

Section: Discussionmentioning

confidence: 99%

“…To measure this, we used bioluminescent imaging (BLI), which is a method that in recent years, is increasingly being utilized to determine location and strength of luciferase activity in a variety of animal models. [47][48][49][50][51] Luciferase activity is observed following an IP dose of D-luciferin, which results in bioluminescence that is detected in anesthetized mice by a cooled charge-coupled device camera. BLI is now an established method for quantitatively detecting transgene expression in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…BLI is a modern transgene expression imaging technique from which the results strongly correlate to transgene expression results measured using traditional tissue and organ harvesting methodologies. [47] We also compare the transgene expression generated by linear and branched PEI-pDNA nanoparticles in a series of cell lines that are commonly used as transfection hosts for evaluating non-viral vector efficiency. Finally, we compare the transgene expression generated by branched or linear PEI-pDNA nanoparticles in vitro and after IP injection in vivo.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

Intra¹,

Salem²

2008

Journal of Controlled Release

124

119

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Polyethylenimine (PEI) is a cationic polymer that has shown significant potential for delivering genes in vitro and in vivo. Mixing cationic PEI with negatively charged plasmid DNA (pDNA) results in the spontaneous electrostatic formation of stable nanoparticle complexes. The structure of PEI can be branched or linear. In this study, we show that branched PEI has a stronger electrostatic interaction with pDNA than linear PEI, which accounts for greater compaction, higher zeta potentials and smaller nanoparticle sizes at equivalent pDNA concentrations. For both linear and branched PEI, increasing the concentration of pDNA mixed in the same volume and at the same nitrogen to phosphate (N:P) ratio results in larger average particle sizes. Increasing the N:P ratio increases luciferase activity generated by branched PEI-pDNA nanoparticles and linear PEI-pDNA nanoparticles in HEK293, COS7 and HeLa cell lines. Increasing the N:P ratio at which branched PEI-pDNA nanoparticles are prepared also increases luciferase expression in HepG2 cells but does not increase luciferase expression generated by linear PEI-pDNA nanoparticles. In all of the cell lines, branched PEI-pDNA nanoparticles prepared at N:P ratios of 10 and above generated significantly higher luciferase activity than linear PEI-pDNA nanoparticles. Luciferase activity was highest in the HEK293 cells and luciferase expression in each of the cell lines followed the order of HEK293

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

Intra¹,

Salem²

2008

Journal of Controlled Release

124

119

View full text Add to dashboard Cite

show abstract

“…[5][6][7][8] According to the results of one of these studies, viral promoters were found to promote the highest initial levels of transgene expression but subsequently expression declined sharply. Expression levels of the initially most effective of these vectors containing a cytomegalovirus (CMV)/ chicken-b-actin fusion promoter element dropped subsequently 31 000-fold within 2 months.…”

Section: Introductionmentioning

confidence: 99%

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

et al. 2008

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An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo

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“…The luminescence in liver was quantified (photons/sec/cm 2 /seradian) by Living Image software and is presented with a pseudo-color overlay of luminescence intensity on grayscale images. The photon intensity in liver was converted into pg of luciferase via a standard curve that was reported previously (41).…”

Section: Dna Formulation Biodistribution and Gene Expressionmentioning

confidence: 99%

Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery

et al. 2007

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PEGylated glycoproteins (PGPs) were synthesized by co-polymerizing a Cys-terminated PEGpeptide, glycopeptide and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20%-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE, and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 hour. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 μg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 hours. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.

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Quantitative bioluminescence imaging of transgene expression in vivo

Cited by 48 publications

References 17 publications

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery

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