Induction of DNA Double-Strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells

Carney, James; Morgan, William F.

doi:10.1007/978-1-4612-1608-7_38

Cited by 2 publications

References 24 publications

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Electroporation

Nickoloff¹

2010

Encyclopedia of Life Sciences

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Electroporation is a simple, rapid and efficient technique for introducing DNA, RNA, proteins and other bioactive molecules into cells and tissues. Electroporation involves the creation of transient, electrically induced membrane pores through which molecules pass into cytoplasmic and nuclear compartments. Because electroporation involves a physical process acting on cell membranes, it is effective with a wide range of cell types including bacterial, fungal, plant and animal cells. Electroporation is used to transiently or stably increase gene expression through transfection of plasmid‐borne gene expression cassettes or decrease gene expression using antisense RNA, small interfering RNA or short hairpin RNA systems. Recent advances have led to new techniques for tissue and organ electroporation in living animals. These techniques are increasingly used in clinical applications including gene therapy, cancer drug delivery and vaccination. Key Concepts: Gene transfer is fundamental to molecular genetic analysis in bacteria, plant and animal cells. Electroporation introduces a wide variety of bioactive molecules into cells, including DNA, RNA, siRNA, proteins, drugs and dyes. Electroporation creates transient membrane pores through which molecules pass into cytoplasmic and nuclear compartments. Electroporation is used to transiently or stably upregulate or downregulate gene expression. Electroporation is used to make stable genetic changes to chromosomes including gene knockout and knockin. Electroporation of molecules into tissues and organs of living organisms is driving novel clinical applications including gene therapy, drug delivery and vaccination. The cytotoxic effects of electroporation are being used in tissue ablation including direct killing of tumours.

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Electroporation

Nickoloff¹

2010

Encyclopedia of Life Sciences

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Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases

Evdokimova¹,

Gandhi²,

Rayapureddi³

et al. 2012

Endocrine-Related Cancer

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Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100 U) or StuI (100 and 250 U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

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Induction of DNA Double-Strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells

Cited by 2 publications

References 24 publications

Electroporation

Electroporation

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases

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