Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter.
Branched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3-6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of b-oligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.
Olsen
P A
PA
Section for Genetic Therapy, Institute of Microbiology, The National Hospital, Oslo, Norway.
McKeen
C
C
Krauss
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D005260:Female; D005434:Flow Cytometry; D005785:Gene Conversion; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D020287:In Situ Nick-End Labeling; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins