Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells

Kamiya, Hiroyuki; Yamazaki, Jumpei; Harashima, Hideyoshi

doi:10.1038/sj.gt.3301831

Cited by 23 publications

(16 citation statements)

References 24 publications

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“…Both the size and topology of the DNA construct were previously described to influence expression of the transgene [20]. In addition, conjugation of an NLS peptide to one of the hairpins was described to further improve transgene expression of the linear DNA constructs both in vitro and in vivo [11,17,19,21].…”

Section: Discussionmentioning

confidence: 99%

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

et al. 2005

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Section: Discussionmentioning

confidence: 99%

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

et al. 2005

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“…The existence of an inverse correlation between DNA size and transfection efficiency has been corroborated by recent results. [15][16][17] Still, a well-assessed interpretation is lacking. Whatever the involved mechanism, it is now well established that all transfection barriers are strongly affected by the ultrastructural features of DNAbased complexes.…”

mentioning

confidence: 99%

“…Such linear DNA is mimetic of plasmid-derived linearized DNAs that are often more efficient than their circular counterpart. 15 We used ultra-tip-sonication to produce DNA fragments between 500 and 3000 bp (tipDNA). Alternatively, DNA fragments longer than 3000 bp (bathDNA) were produced by ultrasound bath sonication.…”

mentioning

confidence: 99%

Nanoscale structure of protamine/DNA complexes for gene delivery

Motta

Brocca

Favero

et al. 2013

Applied Physics Letters

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Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

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“…However, PCR-amplified DNA has not been utilized generally in gene expression experiments in mammalian cells. Although there are many reports of gene expression experiments using restriction enzyme-digested, linearized, plasmid DNA [38][39][40][41][42][43][44], direct utilization of PCR-amplified DNA is not common. Suzuki et al [45] and Hoat et al [46] utilized PCR fragments in high-throughput gene expression experiments using a sensitive luciferase assay from PCRamplified DNA.…”

Section: Discussionmentioning

confidence: 99%

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

et al. 2015

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Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

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Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells

Cited by 23 publications

References 24 publications

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

Nanoscale structure of protamine/DNA complexes for gene delivery

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

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