Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.
We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.
Sommer
Jeffrey R
JR
Department of Animal Science, North Carolina State University, Raleigh, NC 27695, USA.
Alderson
Jon
J
Laible
Goetz
G
Petters
Robert M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002999:Clone Cells; D003116:Color; D006224:Cricetinae; D003412:Cricetulus; D005785:Gene Conversion / Q000235:genetics*; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D008164:Luminescent Proteins; D009841:Oligonucleotides; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins