Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.
Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.
Schagen
F H
FH
Departments of Molecular Cell Biology and Biochemistry, Leiden University, Leiden, The Netherlands.
Rademaker
H J
HJ
Cramer
S J
SJ
van Ormondt
H
H
van der Eb
A J
AJ
van de Putte
P
P
Hoeben
R C
RC
D010583:Bacteriophage mu / Q000235:genetics; D002461:Cell Line, Transformed; D004251:DNA Transposable Elements / Q000235:genetics; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019895:Transposases / Q000235:genetics; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C069215:MuB protein, Enterobacteria phage Mu; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C085706:FLAG peptide; C114697:mu transposase; D019895:Transposases