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Silencing of retroviral vector transduced LacZ reporter gene by frameshift mutation
Author(s) -
Valkova Christina,
Georgiev Oleg,
Karagyozov Luchezar,
Milchev Georgi
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10715
Subject(s) - biology , transduction (biophysics) , frameshift mutation , microbiology and biotechnology , lac operon , gene , transposable element , reporter gene , viral vector , mutation , plasmid , open reading frame , gene silencing , genetics , gene expression , mutant , recombinant dna , peptide sequence , biochemistry
Moloney murine leukemia virus‐based vector expressing Escherichia coli β‐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 β‐galactosidase positive or β‐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 β‐galactosidase positive as well as in one of three investigated β‐galactosidase negative cell lines. DNA sequence analysis of proviral LacZ gene in β‐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 β‐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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84:1–6, 2003.