Transposons: cut-and-paste gene delivery

TECHNOLOGY FEATURE Jumping in the tool box 183 Off-the-shelf transposition 184 The therapeutic horizon 185 Transposons for the lab menagerie 185 © 2007 Nature Publishing Group http://www.nature.com/naturemethods Transposons: cut-and-paste gene delivery From mutagenesis to gene therapy for hemophilia, transposons⎯mobile genetic elements⎯have proven themselves innovative tools in the laboratory and the clinic. Caitlin Smith takes a look at some present offerings of transposon products and the promise of applications. Gene therapy, the therapeutic culmination of years of research on gene targeting and delivery, involves the ability to deliver a gene to the relevant organ so that it can correct a naturally corrupted genetic function. The technical difficulties of such achievement are not limited to therapeutic promises. The delivery of exogenous genetic material, by no means easy despite recent advances, is an invaluable tool for researchers needing to introduce genes into cell lines, tissues in culture or even whole animals for research purposes. Research tools and therapeutic promises are evolving in parallel, and although gene therapy cannot yet be considered as routine clinical procedure, gene delivery and targeting in experimental systems is a ubiquitous tool in many research labs. Jumping in the tool box Among the most important methods used for gene delivery are viral vectors, which take advantage of the natural ability of viruses to infect a host’s cells, and RNA transfection, a newer tool that permits the delivery of mRNA or small noncoding RNAs. Many companies have invested in the development and refinement of such tools for gene delivery, and researchers have a panoply of reagents at their disposal. In addition to these reagents, researchers and companies alike have recently turned toward another promising tool: mobile genetic elements called transposons. Transposons, also known as jumping genes, are sequences of DNA that can move from one site in a genome to another by the process of transposition⎯the 1983 Nobel prize–winning work of Barbara McClintock. Transposition has the potential to wreak havoc in a genome: it can result in lethal mutations and can alter the amount of DNA in the genome. Yet surprisingly, we apparently have learned to live with it. The human genome contains more than 3 million copies of transposons, which comprise about 45% of our total DNA, with each transposon copy separated by only 500 nucleotides on average. Certain transposons, however, can cause diseases, including hemophilia A and B, severe combined immunodeficiency, porphyria, predisposition to cancer and Duchenne muscular dystrophy. Researchers’ abilities to manipulate transposons, to use them as tools for gene insertion and delivery, is steadily growing. One well-known family of Drosophila melanogaster transposons, called P elements, appeared in the species in the middle of the twentieth century and spread through every D. melanogaster population within 50 years. These have proven useful to researchers because P elements can be injected into embryos to insert genes into D. melanogaster. The occurrence of transposons in many different organisms is an exciting aspect of their usefulness as research tools. According to David Largaespada, associate professor in the department of genetics, cell biology and EZ-Tn5 in vitro insertion reaction Target DNA DrugR EZ-Tn5 transposon EZ-Tn5 transposase 1. Incubate 37 °C; 2 h 2. Transform E. coli 3. Select drug-resistant clones 4. Prepare template DNA Sequence bidirectionally from primer binding sites ( ) The process for generating DNA sequencing template using an Ez-Tn5 Insertion kit. (Courtesy of Epicentre Biotechnologies.) NATURE METHODS | VOL.4 NO.2 | FEBRUARY 2007 | 183 Entranceposon (STOP) MuA Transposase Target DNA with a gene of interest In vitro transposition reaction (incubate 60 min at 30 °C, heat inactive 10 min at 75 °C) TO P OP 3× ST Result: library of truncated proteins ready for expressions studies COOH Target DNA clone NH2 3× STOP 3× STOP COOH STOP Clone 1 NH2 3× STOP COOH 3× STOP STOP Clone 2 NH2 3× STOP 3× STOP STOP Clone 3 NH2 H O 184 | VOL.4 NO.2 | FEBRUARY 2007 | NATURE METHODS 3× STOP O formation of a tetramer of MuA transposase around the terminal sequences of a linear Entranceposon DNA molecule. The activated transposon-complex finds and makes a 5-base-pair staggered cut in the target DNA, and then the Entranceposon DNA is inserted into the target site. One advantage of kits that use the enzyme MuA transposase, such as those from Finnzymes and other manufacturers, is that the MuA-catalyzed in vitro transposition is essentially random, so there are no hot-spots for insertion. As Tieaho explains, “Some other in vitro transposition systems tend to favor, for instance, (A+T)rich areas in target DNA over (G+C)-rich areas.” Like the Finnzyme Template Generation System II, Invitrogen also offers a kit aimed at DNA sequencers. Their GeneJumper Kit is designed to insert primer binding sites randomly into target DNA in preparation for sequencing. Each kit includes the GeneJumper Transposon with chloramphenicol or kanamycin resistance, MuA Transposase, reaction solutions, PCR and sequencing primers, control template and primers, One Shot competent Escherichia coli, SOC medium and a control plasmid. New England BioLabs’ GPS-1 Genome Priming System is another such kit for researchers who want to generate a population of DNA sequencing templates with randomly interspersed primer-binding sites. GPS-1 is a transposon-based in vitro system that uses the TnsABC Transposase to insert a transposon (NEB’s Transprimer) randomly into the target DNA. The GPS-1 System also includes two Transprimer transposons, encoding kanamycin or chloramphenicol resistance. Epicentre offers too wide a selection of transposon-based products to detail here, but they generally fall under the headings of the Epicentre EZ-Tn5 Transposon Systems and HyperMu Transposon Systems, which are designed for both in vitro and in vivo applications. Among their many offerings are kits and products for sequencing applications like others, but also for making unidirectional truncations of a protein encoded by any sequence, mapping protein domains In vitro transposition reaction components C Off-the-shelf transposition Several companies offer transposon-based kits for just these types of projects. Finnzymes, for example, offers their Finnzymes Transposon Tool product line, based on the in vitro transposition reaction of the bacteriophage Mu, a common transposon tool. These tools are packaged into three separate transposonbased kits that suit different applications: the Template Generation System II introduces primer binding sites randomly into foreign target DNA, the Mutation Generation System generates insertion mutation libraries from any target gene, and the Stop Generation System inserts translational stop codons into any target gen (...truncated)