Journal: Nucleic Acids Research
Article Title: Site-directed transposon integration in human cells
Figure Lengend Snippet: Site-directed transposition in human cells. ( A ) Schematic overview of plasmid-based assay for investigating site-directed transposition. Transposition was initiated by transfecting human HeLa cells with a plasmid encoding chimeric or unfused HSB5 transposase, together with a donor plasmid encoding a bleomycin-marked (zeo r ) transposon and a counter-selectable chloramphenicol-resistance (cam r ) gene. These plasmids were co-delivered with an ampicillin-resistant (amp r ) target plasmid containing five tandem DBD recognition sequences and allowed to undergo transposition. Low-molecular weight plasmid DNA fractions were isolated 2 days later and transformed into DH10B E. coli . Replication of the R6K origin-containing donor plasmid is strictly dependent on expression of the pir1 gene product, which is absent in this bacterial strain. Amp r /zeo r bacterial colonies were patched onto LB-cam r plates to screen for inter-plasmid transposition events specific for the target plasmid (i.e. cam s ). Both pooled and clonal amp r /zeo r /cam s populations of bacteria were amplified, plasmid DNA isolated and the locations of transposon insertions relative to the target sites determined by restriction site analyses and DNA sequence analyses, respectively. ( B ) Target plasmid features. Positions of BglI and BglII restriction endonuclease recognition sites are shown, as are the sizes for each resulting DNA fragment. ( C ) Southern blot analysis of targeted integration. For each experimental condition, 500 ng of plasmid DNA isolated from pooled amp r /zeo r /cam s bacterial colonies ( n = 43–51) was treated with BglI-BglII restriction enzymes. Samples were resolved on an agarose gel, transferred to nitrocellulose, hybridized to a 32 P-radiolabelled probe corresponding to the left SB transposon inverted repeat, and resulting bands visualized upon autoradiography. In one instance, excess E2C DNA-binding domain was co-expressed with transposase protein to determine whether associated proteins could inhibit SB target site DNA capture. The lower band intensity under each experimental condition represents a qualitative assessment of the relative frequency of transposition of the 1.35 kb zeo r -marked element into the 443 bp targeting window. ( D ) Targeted transposition frequencies. Recombinant target DNA molecules were isolated from individual amp r /zeo r /cam s colonies and sequenced using an internal transposon-specific primer. Bars denote the percentage of total integrations occurring within the 443-bp targeting window, whereas the numbers in parentheses denote the actual number of integrations analyzed in each group. ( E ) Flexing model for targeted transposition. Multiple changes in both DNA- and protein conformation are necessary to complete a fullcycle of transposition. A protein that remains too tightly bound to DNA, such as hE2C-L5-SB to the canonical e2c site, cannot efficiently catalyze these reactions. In the case of the mutant e2c site, however, only fingers 1 through 3 of hE2C-L5-SB retain the capacity for DNA-binding. This would essentially improve the flexibility of the transposase domain, which might enhance the acquisition and/or manipulation of neighboring target sites.
Article Snippet: PCR products were ligated to the pGEM-T easy vector (Promega), transformed into DH10B cells (Invitrogen) and analyzed using the IR-1 sequencing primer.
Techniques: Plasmid Preparation, Chick Chorioallantoic Membrane Assay, Molecular Weight, Isolation, Transformation Assay, Expressing, Amplification, Sequencing, Southern Blot, Agarose Gel Electrophoresis, Autoradiography, Binding Assay, Recombinant, Mutagenesis