© Dr. Jeremy Burgess/Science Photo Library
If you thought transposons were mere genetic curiosities, think again. In the hands of research scientists, genetic elements akin to those that give Indian corn its unique coloration can be used to sequence genes and genomes, create libraries of mutants in vitro, and even make mutations in vivo.
Geneticists have been tinkering with the
A transposon, or "jumping gene," is simply a gene coding for a transposase (an enzyme that catalyzes excision and insertion of the DNA between specific recognition sequences) sandwiched between two copies of the enzyme's recognition sequence – the epitome of minimalist functionality....
BUILDING SEQUENCING LIBRARIES
When molecular biologist Richard McCombie of Cold Spring Harbor Laboratory wanted to put the finishing touches on the rice genome sequence, he turned to NEB's Genome Priming System (GPS)-1, which uses the ends of randomly inserted transposons as sequencing primer-binding sites.
"We thought it would be easier, with the robotics we had, to adapt transposons for high throughput than to use oligos," says McCombie. "The nice thing about transposons is that you can identify your clones and for a reasonable cost can sequence the entire clone. In some cases, too, transposon insertions disrupt secondary structure that might interfere with [sequencing] .... We are still using [GPS-1] to finish some of the really difficult clones in the rice genome."
McCombie notes, however, that for financial reasons, he is "planning on looking at primer walking again." Primer walking involves sequencing with a custom primer, designing another custom primer near the end of the new sequence, and repeating as needed. It is the clear choice for short templates, but for projects involving more than 2 kb, transposons are an attractive alternative, says McCombie. "As oligos keep getting cheaper and cheaper and have a sharper downward [price] curve than transposon systems, at some point they may become more attractive."
According to Fred Hyde, technical product manager at Epicentre, typical transposition reactions yield hundreds or thousands of insertions, so one reaction per clone is usually sufficient. For instance, to sequence an 8-kb template with 1x coverage, "you're ... picking 10 to 12 clones," he says. Epicentre and NEB kits are similarly priced at about $25/reaction; Finnzymes' cost $10/reaction.
GENERAL MUTAGENESIS
Any transposon kit can create libraries of mutations in vitro, but several are designed specifically for this purpose. "With transposon-based mutagenesis systems, you can generate saturated libraries of mutated proteins for functional analysis," says Arto Forsberg, Finnzyme's international sales director.
Nina Salama, a microbiologist at the Fred Hutchinson Cancer Research Center in Seattle, modified NEB's GPS-M system to mutagenize
Finnzyme's STOP Kit and NEB's GPS-M Kit insert transposons containing stop codons in all frames, creating libraries of C-terminal deletions; Epicentre's EZ-Tn5 Protein Truncation Kit and companion Mu-End Protein Truncation Kit use a more complex scheme to truncate proteins, creating libraries of both N- and C-terminal mutations.
Some kits, including NEB's GPS-LS, include strategic restriction sites so that the transposon can be trimmed to a smaller "linker-scanning" type of mutation. In the case of GPS-LS, the result is insertion of five amino acids in four reading frames and stop codons in two; Finnzymes' and Epicentre's kits use similar methods to insert five and 19 amino acids, respectively, in all frames. Kits range from $20 to $31 per reaction.
BUILD YOUR OWN
All three transposon manufacturers make components available individually to facilitate custom transposon construction. Microbiologist Cindy Arvidson of Michigan State University, for instance, used a custom transposon based on Epicentre's pMod-2<MCS> vector to mutagenize plasmids encoding possible cytoplasmic membrane proteins in
The construct, which was designed by Brian LeCuyer, a technician working with microbiologist Hank Seifert of Northwestern University, includes an antibiotic resistance gene and a
Epicentre also offers in vitro kits with functions beyond mutagenesis and primer insertion. The company's EZ-Tn5 <R6Kγori/KAN-2> and EZ-Tn5 <oriV/KAN-2> R6Kyori insertion kits, for instance, incorporate
IN VIVO MUTAGENESIS
Most transposon kits function in vitro. But Epicentre's "transposome" approach works in vivo, at least in prokaryotes. A transposome is a transposon/transposase complex frozen (by the lack of magnesium) in the act of cleavage. These inactive complexes can be electroporated into a wide variety of prokaryotes, where intracellular magnesium activates the enzyme, inducing transposition events in vivo.
According to Hyde, more than 30 papers have been published to date using this approach, including one by Thomas Kawula, a microbiologist at the University of North Carolina, Chapel Hill. He uses transposomes to mutagenize
The EZ-Tn5 Tnp Transposome kit "worked very well for us," Kawula says: The system produced stable, relatively random insertion mutations despite "statements in the literature that transposon mutagenesis wouldn't work in
Mammalian Transposons
Though commercial transposon kits are designed for use in vitro or in prokaryotic cells, researchers also have historically used jumping genes to mutagenize and introduce genes in
Discovery Genomics, based in Minneapolis, is using one such system, called Sleeping Beauty, to develop gene therapy approaches for human use, according to chief science officer Perry Hackett. Sleeping Beauty is also useful for inducing mutations in mammalian model systems. Recently, for instance, there have been "two spectacular papers on the use of Sleeping Beauty to uncover oncogenes in normal mice and mice that are predisposed towards cancer," Hackett says.12
David Largaespada of the University of Minnesota Twin Cities, senior author of one of the articles,1 says, "The Sleeping Beauty system allows us to randomly mutate genes in cells in mice. Given enough time the insertional mutation will cause cancer, because we designed the vector to activate proto-oncogenes or inactivate tumor suppressor genes. If we use a cancer-predisposed strain of mouse, we can find out what mutations cooperate with the mutation present in that strain to produce cancers."
Another transposon system, piggyBac, has also been shown recently to work in mammals.3 According to molecular geneticist Malcolm Fraser, Jr., at the University of Notre Dame, both "integration and excision of piggyBac are very precise. It targets a TTAA sequence to interrupt, and regenerates the sequence on excision. This way you get the ability to do forward genetics [create mutations] and can do reverse genetics very nicely by just removing the transposon."
Though neither system has been commercialized, both are readily available. Academic and commercial groups can utilize Sleeping Beauty by signing a material transfer agreement (MTA) with the University of Minnesota. Academic researchers can obtain piggyBac by completing an MTA from the University of Notre Dame
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