Silencing of retroviral vector transduced LacZ reporter gene by frameshift mutation.
Moloney murine leukemia virus-based vector expressing Escherichia coli beta-galactosidase (lacZ) as reporter gene and the transposon Tn5 neomycin resistance (neo) gene was transduced at low-multiplicity of infections into NIH 3T3 cells. Geneticin (G418)-resistant cells were recloned and cell lines containing beta-galactosidase positive or beta-galactosidase negative cells were obtained. Both positive and negative cell lines contained a single proviral copy at distinct integration sites. RNA complementary to lacZ was detected in beta-galactosidase positive as well as in one of three investigated beta-galactosidase negative cell lines. DNA sequence analysis of proviral LacZ gene in beta-galactosidase negative cell line C6 showed a single nucleotide insertion at position 1567 resulting in reading frame shift and translational stop codon at position 1629. This mutation explains the enzyme inactivation. The absence of beta-galactosidase after retroviral transduction of LacZ reproter gene may be a consequence of definite mutation but not a consequence of ineffective transduction or transcriptional inactivation of transgene.
Valkova
Christina
C
Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria.
Georgiev
Oleg
O
Karagyozov
Luchezar
L
Milchev
Georgi
G
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D016368:Frameshift Mutation / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D020868:Gene Silencing / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001616:beta-Galactosidase