Optimisation of Electroporation for Biochemical Experiments in Live Cells

Meldrum, Rosalind A.; Bowl, Michael R.; Ong, Swee Bee; Richardson, Simon C. W.

doi:10.1006/bbrc.1999.0246

Cited by 29 publications

(24 citation statements)

References 11 publications

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“…This makes them unsuitable for introduction of plasmids that can alter cellular activities (63)(64)(65). However, they take up DNA oligonucleotides without treatment such as lipofection or electroporation that can destroy cellular membranes.…”

Section: Discussionmentioning

confidence: 99%

Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein β Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells

Ji¹,

Studzinski²

2004

Cancer Research

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Section: Discussionmentioning

confidence: 99%

Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein β Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells

Ji¹,

Studzinski²

2004

Cancer Research

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“…The experimentally useful ability of strain 98/2 to permit site-directed gene replacement through homologous recombination could be the result of the observed mild but persistent upregulation of DSB repair protein transcripts following the application of a DNA damaging agent, in this case UV-C irradiation. DNA uptake by S. solfataricus is facilitated by electroporation, a transformation method that has been reported to cause cellular damage by inducing DNA breaks (26,27). Assuming similar DSB repair protein stability in vivo in Sulfolobus, it may be that the ease with which strain 98/2 performs directed gene replacement using exogenous DNA is the result of persistent DSB repair pathway induction that would give cells an extended opportunity to accomplish homologous recombination.…”

Section: Discussionmentioning

confidence: 99%

Repair of DNA Double-Strand Breaks following UV Damage in ThreeSulfolobussolfataricusStrains

2010

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DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for doublestrand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.Cells have evolved molecular mechanisms to meet the challenge of maintaining genomic integrity by rapidly responding to environmental stresses that can damage proteins and DNA. One of the most common forms of damage is caused by UV light (UV) exposure. High-energy short-wavelength UV-C light is absorbed directly by DNA and induces both cyclobutane pyrimidine dimers between adjacent thymidine or cytosine residues as well as pyrimidine-pyrimidone photoproducts between adjacent pyrimidine residues. Mechanisms for repair of these lesions appear to be present in all organisms and are thought to occur through either light-independent nucleotide excision repair (NER) or light-dependent photoreactivation using photolyases (for reviews, see references 33 and 34). UV-C irradiation also causes the production of reactive oxygen species, which can result in DNA double-strand breaks (DSBs) (6, 44). Our primary understanding for repair of DSBs has come from studies focused primarily on bacteria and eukaryotes. In Escherichia coli, these breaks are repaired through the action of the RecA protein, which assists in recombinational repair of single-strand regions produced through replication fork arrest at UV lesions and in DSB repair by extended synthesis-dependent strand annealing (SDSA) and homologous recombination (9, 19). Eukaryotes employ nonhomologous end joining as well as DSB repair by SDSA and homologous recombination mechanisms to repair these breaks (for recent reviews, see ref...

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“…162,163 The need to optimize gene transfer efficiency by electroporation is somewhat less complex than the optimization of a lipoplex formulation as only two important electrical parameters determine, at least to a great extent, the degree of physical gene transfer. 164,165 The most important parameter is the electric field strength E determined by the formula E = V/d, where V is the applied voltage and d is the distance between the electrodes of the cuvette (usually 0.4 cm). The higher the voltage, the more and the larger pores will be formed, although cell toxicity will increase with increasing voltage.…”

Section: Electroporationmentioning

confidence: 99%