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Gregory Wagner

Lec 23 BIOL207 2014-03-07 A. Transgenic organisms a. introduction of “foreign” DNA into an organism (e.g. different species, or recombinant version of DNA from same species). i. foreign DNA is usually a complete expression unit, i.e. protein coding genes and regulatory sequences (e.g. promoters) ii. foreign DNA usually also includes a selectable marker (e.g. antibiotic resistance gene) and its promoter, so that non-transgenic cells can be eliminated b. making a transgenic cell … is called transformation (bacteria, fungi, plants) … is called transfection (animals) c. minimal requirements i. transfer of DNA across cell & nuclear membrane DNA may be “naked” or attached to a carrier methods of transfer: freeze/thaw (especially bacteria) CaCl 2especially bacteria) electroporation (especially bacteria, yeast; some animals) microinjection (especially with animal cells) engineered viruses (especially with animal cells) engineered bacteria (especially with plant cells) particle bombardment (especially with plant cells) ii. once in nucleus, transgenic DNA may or may not be inserted into chromosomal DNA 1. if not inserted into chromosomes: usually leads to transient transformation (i.e. not heritable) 2. if transgene inserted into chromosomes: usually leads to stable transformation a. this is usually heritable IF germline is transformed b. insertion may occur at a random location c. insertion may be targeted to a particular location i. depends on homologous recombination so foreign DNA must be flanked by DNA with high of similarity to target locus ii. targeted insertion works in animals & fungi, but not in most plants B. Making a transgenic plant using Agrobacterium-mediated transformation a. vector: “T-DNA plasmid”
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