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Stable integration of large PAC constructs in keratinocytes.

Published on Jan 1, 2005in Methods of Molecular Biology
路 DOI :10.1385/1-59259-830-7:315
Sarah H. Williams2
Estimated H-index: 2
(University of Oxford),
Alain Hovnanian38
Estimated H-index: 38
Abstract
Transfer of P1-derived artificial chromosome (PAC) deoxyribonucleic acid (DNA) into keratinocytes is an extremely important technique that enables functional studies of keratinocyte-specific genes to be performed and genomic gene therapy for inherited and acquired diseases to be attempted. Ex vivo gene therapy approaches are possible using well-established conditions for keratinocyte culture and grafting, whilst the skin is the most accessible organ for administering in vivo therapy. PAC vectors lack relevant reporter genes to distinguish transfected mammalian cells from the non-transfected background, or to select clones in which the PAC construct has stably integrated into the genome. In this chapter, protocols to retrofit a reporter gene cassette will be described, together with techniques for transfecting large PAC constructs into keratinocytes without breakage. Protocols to select for stable integrants and to assess the integration event(s) within the keratinocyte genome will also be provided.
  • References (11)
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References11
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#1S H Compton (Wellcome Trust Centre for Human Genetics)H-Index: 1
#2Sabine Mecklenbeck (WWU: University of M眉nster)H-Index: 4
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A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage s...
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We have designed a P1 vector (pCYPAC鈭1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1鈭抎erived arteficial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130鈭150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that...
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