Use of epitope tags for routine analysis of transgene expression.
Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.
Alarcon
C M
CM
Trait and Technology Development, Pioneer Hi-Bred International, Johnston, IA 50131, USA.
Umthun
A R
AR
Register
J C
JC
D000123:Acetyltransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019470:Biolistics; D015153:Blotting, Western; D002645:Chickens; D000939:Epitopes / Q000235:genetics* / Q000276:immunology*; D005658:Fungi; D005692:Galactose Oxidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007118:Immunoassay; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013302:Streptomyces; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*; D003313:Zea mays / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D000939:Epitopes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001360:Avidin; D005692:Galactose Oxidase; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase