TECHNOLOGY FEATURE

Set your sites on mutants 458 Recombineering: how does it work really? 458 Manipulating the bad bugs 459 Box 1: Gene trapping for the future 455 Box 2: Neuromutagenesis centers 456 Cloning and mutagenesis: tinkering with the order of things Engineering the replication of target DNA through cloning, or changing its genetic code through mutations, are detail-oriented processes whose foibles can spell disaster. Caitlin Smith looks at some new tools and techniques that may smooth the road to a successful experiment.

‘Clone the cDNA from the cells under treat- ment, insert it into our specialized expression BOX 1 GENE TRAPPING FOR THE FUTURE vector, and mutate regions x, y and z, which we suspect are important for the gene’s Gene trapping is a high-throughput approach to making insertional mutations in function’—this list of instructions could mammalian genomes. It is useful because gene traps simultaneously inactivate have been a 5-year thesis project 20 years and report the expression of the ‘trapped’ gene at the insertion site, and provide a ago. Today, it is the likely weekly program DNA tag for the rapid identification of the disrupted gene. Patricia Ruiz Noppinger of many laboratory technicians. Cloning from the department of vertebrate genomics at the Max Planck Institute for and mutagenesis, once revolutionary tech- Molecular Genetics, uses gene trapping in her lab in a large-scale generation of niques that launched the molecular biology mutant embryonic stem cell lines. Noppinger says that “the most challenging era, have now become common techniques aspect of learning to use gene trapping is the analysis and understanding of the no longer constrained to molecular biology mutation created by the gene trap vector, which includes the determination of labs. The evolution of these basic tools has the exact integration site of the vector at the genomic level.” The method is not been spurred by developments brought to without limitations. It has been generally assumed that only about 50% of mouse the market by many companies. With the genes may be mutated using gene trapping, though Noppinger’s lab has evidence myriad of kits available, it seems that clon- that trapping can actually reach up to 70–80% of targetable genes. Additionally, ing and mutagenesis are becoming faster and gene trapping leaves behind cassettes that could have unknown and potentially easier every day. harmful effects on the mutant phenotype. A recent advance that is proving to be highly William Stanford, director of Gene Trap Mutagenesis at the Centre for Modeling efficient and relatively simple is recom- Human Disease at the University of Toronto agrees that “one limitation is that bineering, now a true alternative to con- many genes have still not been trapped by any of the labs of the International ventional cloning. Though originally Gene Trap Consortium (IGTC).” The result of the work in labs participating in the observed in Escherichia coli, recombineering IGTC is that more than 60% of the genome has been trapped. “There are hot spots seems to export quite well to other model for trapping and some genes are refractory to gene trapping,” explains Stanford. organisms. The technique, whose fine mech- “Thus, some groups have abandoned gene trapping as a mutagenesis tool and are anisms are still not completely understood, is now performing high-throughput gene targeting. Our poly(A) trap resource has attracting increasing attention as a potential a 70% unique gene trap hit rate, and thus we are still generating new mutations solution for manipulating the genome of at a high rate. However, we are part of a large consortium, the North American organisms for which there are fewer tools Conditional Mouse Mutagenesis Project, which is using high-throughput gene than for E. coli. targeting to generate conditional mutagenesis alleles in 2,000 genes.” Other methods of mutagenesis have As for the future of gene trapping, Stanford believes that it still has promise also enjoyed increasing popularity with for cell lines other than mouse embryonic stem cells⎯such as human embryonic the advent of large-scale projects to gener- stem cells and other cell lines, or even for use in mutagenesis of other species. ate collections of mutant mouse lines. The There are presently several gene trap screens being performed in non-murine International Gene Trap Consortium, for model organisms. example, as well as other smaller research Noppinger believes that using conditional gene trap vectors together with groups, are focusing on systematically the ability to determine the insertion sites at the genomic level (for example, mutating all genes in the mouse using gene by splinkerrette PCR) will greatly affect gene trapping in the future. “Moreover, trapping (Box 1). Several groups are also gene trapping cassettes are currently being designed that will allow recombinase- focusing on thematic mutagenesis, system- mediated cassette exchange. A further aspect we are thinking about is the atically knocking down all genes related to a potential use of gene trap vectors for gene therapy in the human system.” given pathway (Box 2).

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Copying, cutting and pasting: marketing at Stratagene. She explains that offers their Phusion High-Fidelity DNA fast-tracks for the old-fashioned way conventional reverse transcriptases typically polymerase and Phusion Hot Start DNA With increasing ease and sophistication, have considerably high error rates, introduc- polymerase. “These polymerases have an developments of PCR and other tools have ing errors at frequencies of one per 1,500– extremely low error rate, 50-fold lower than put processes that used to be time-consum- 30,000 nucleotides during cDNA synthesis. that of Thermus aquaticus DNA polymerase ing for skilled molecular biologists into every In contrast, Stratagene’s Accuscript RT, says and sixfold lower than that of Pyrococcus scientist’s hands. The PCR products of today Formosa, “contains a 3′-to-5′ proofreading furiosus DNA polymerase,” says Netta Fatal, are more accurate replicates of their tem- exonuclease, which reduces errors three- to marketing communications manager at plates, and there is a kit for nearly every pos- sixfold during first-strand cDNA synthesis.” Finnzymes. sible way of cloning a DNA fragment (from For the next step of cloning cDNA or other In principle, cloning PCR products transcribed RNA or another source) into a DNA fragments, improved PCR enzymes necessitates some fancy footwork on the vector with a specialized function. have been available for a while that improve part of the researcher. But in practice with For a start, PCR has not only made cloning cloning accuracy by reducing the error rates the help of a battery of kits, all you need much easier; improvements to the techno- during PCR. For example, Stratagene offers to know is the nature of the ends of your logy also continue to improve cloning accu- its high-fidelity PfuUltra II Fusion HS DNA fragments⎯staggered, blunt and perhaps racy. “Many people do not realize that reverse polymerase, which according to Formosa containing sites for restriction enzymes. transcription is a major contributor to errors generates “only 1 error per 2.5 million bases. ‘Old-fashioned’ cloning still works, and for when cloning a fragment that originated It is also good for the replication of long tar- years, researchers relied on using cloning from RNA,” says Rachel Formosa, director of gets, up to 19 kb, and requires only 15 seconds methods in which vector and insert were amplification and gene expression product per kb extension time.” Likewise, Finnzymes outfitted with restriction sites to make them

BOX 2 NEUROMUTAGENESIS CENTERS

A few years ago, the US National Institutes of Health funded three “First, the behavioral tests for these phenotypes are inherently projects scattered around the US that had something in common: noisy, and that makes detecting outliers a difficult problem. a focus on neuromutagenesis the mutation of genes involved in Second, as the behavioral readout is not clean, the mapping of the nervous system. The three locations were the Neurogenomics the mutant gene is a problem that can not be solved by Mendelian Project at Northwestern University, the Neuroscience Mutagenesis methods. Mutants need to be mapped by quantitative trait locus Facility at The Jackson Laboratory, and the Neuromutagenesis analytic tools. Third, because of the imprecision of this sort of Project of the Tennessee Mouse Genome Consortium. Researchers mapping, it is more challenging to identify the gene. And we all at all three centers generated mutants lines of mice by randomly need the gene if we want to call this effort a success.” generated mutations using a chemical known as ethylnitroso-urea Joseph Takahashi, Howard Hughes Medical Institute (ENU). To facilitate the sharing of results and the distribution investigator at Northwestern University, heads the Northwestern of the resulting mutant mice with other scientists, the three branch of NeuroMice.org. According to Takahashi, the goal of their groups came together to form a consortium called Neuromice.org, program is “to use forward genetics to isolate mouse mutants for manifest in a common website through which anyone could order neural and behavioral phenotypes. The potential for novel gene mice. Although no longer functioning as a mutant distribution discovery is high because forward genetics makes no assumptions center, the website still acts as a repository for valuable on the underlying mechanisms involved and therefore completely information, and a directory for mutant lines (which can still be unsuspected pathways can be uncovered.” He cites the circadian ordered from the participating centers). clock and obesity mutants as the best examples of the success of Dan Goldowitz’s group at the Tennessee Mouse Genome this approach. Consortium was one of the three cofounders of Neuromice. Making neuromutants is a laborious process. Takahashi org. Goldowitz, a professor in the department of anatomy and describes how they sort mutants and decide which lines to neurobiology at the University of Tennessee Health Science develop. “We produced and screened 10,000 mice per year Center, explains why he chose to focus on neuromutagenesis: and screened over 35,000 mice. We screened using high- “The nervous system is seen as the last biomedical frontier in throughput behavioral screens in which we could automate data several respects, and this is never more clear than in trying to acquisition. Each screen had to have a capacity of more than understand the role of single genes in complex behaviors.” The 200 mice per week (to achieve the goal of 10,000 mice screened goal of Goldowitz’s Neuromutagenesis Project is to mutagenize the per year).” The major screens included fear conditioning, mouse genome and put the mice through sophisticated screens locomotor response to cocaine treatment, motor and balance to identify individual pedigrees to demonstrate neurological deficits, visual defects, circadian activity rhythms and serum phenotypes. “The screens are the sorting mechanism,” says glucose levels (diabetes). Goldowitz, “and unless the mutant had an obvious or a gross Takahashi says that the most difficult aspect of creating phenotype like ataxia or failure to thrive, we designed an neuromutants is the development of a robust behavioral screen. unbiased statistical cut-off to tell us which lines to pursue. Those Although they “have developed new strategies for mapping that met that statistical criteria were further developed.” behavioral mutants to cope with genetic background issues and Goldowitz says that there are still problems with trying to study the variance of phenotypic assays, much more robust behavioral mice with complex phenotypes such as drug-seeking behavior: assays need to be developed for screening.”

456 | VOL.4 NO.5 | MAY 2007 | NATURE METHODS TECHNOLOGY FEATURE compatible. But to avoid additional time- consuming restriction steps, manufacturers 1. Mutant strand synthesis have developed kits with neat tricks to exploit Perform thermal cycling to: X • denature DNA template the generic fragment ends generated by Taq X • anneal mutagenic primers polymerases or convert them to blunt ends containing desired mutation that will clone efficiently. • extend and incorporate primers Many companies offer cloning kits, includ- with PfuUltra DNA polymerase ing Invitrogen, Lucigen, Promega, Epicentre, Clontech and New England BioLabs, among X X others. A relatively new kid on the block, Real Biotech Corporation (RBC) wants to beat the clock. The company offers their RBC T&A 2. Dpnl digestion of template Digest parental methylated and cloning kit, in which vectors that contain hemimethylated DNA with Dpnl inserts will also have two new restriction sites, X so you can tell whether your DNA fragment X has been incorporated. They claim that “the combination of [our] RBC ligation kit and HIT 3. Transformation Competent Cells allow the whole process to be Transform mutated molecule into competent cells for nick repair completed within six minutes,” according to X Joyce Kuo, international sales manager at RBC. X “RBC’s HIT Competent Cells is the fastest transformation system worldwide (completed The QuikChange II one-day site-directed mutagenesis method. Mutant-strand synthesis is followed by DpnI digestion of the parental DNA template, and transformation of the resulting annealed double- in less than one minute) and can be applied to stranded nicked DNA molecules. After transformation, the XL-1 Blue E. coli cell repairs nicks in the all common E. coli laboratory strains.” plasmid. (Courtesy of Stratagene.)

Set your sites on mutants Site-directed mutagenesis has become so sion made specifically for the electropora- expressed and regulated so as to maximize valuable an experimental tool that several tion-competent-cell users, as well as a ver- recombination efficiency. companies now offer kits to streamline the sion to introduce multiple point mutations at Donald Court, senior investigator at process. Consider Finnzymes’ Phusion Site- different sites simultaneously,” says Formosa. the Center for Cancer Research at the US Directed Mutagenesis kit for making point Other companies offering site-directed muta- National Cancer Institute in Frederick, mutations, insertions or deletions in plas- genesis kits include Clontech, Invitrogen, Maryland, USA is one of the pioneers of mid DNA. “The use of Phusion Hot Start Promega and Takara Bio. the technique and an enthusiastic user. He DNA polymerase in amplification guaran- says: “our laboratory uses recombineering tees extreme fidelity and specificity to the Recombineering: how does it work really? for all types of purposes, including gene reactions,” says Fatal. “No special vectors or Recombineering, or recombinogenic knock-outs, gene knock-ins, gene fusions, restriction sites are required, and also large engineering, is a powerful cloning and muta- gene point mutations⎯both directed to a plasmids (up to 10 kb) can be used. The genesis technique based on homologous specific nucleotide or randomly generated mutagenesis protocol comprises only three recombination in E. coli. Several systems throughout the gene⎯gene cloning, plasmid steps, making it simple and easy to perform.” have been developed that take advantage of and bacteriophage constructions.” A particu- For generating large libraries of mutated pro- phage proteins to efficiently manipulate DNA larly useful application of this technology is teins, Finnzymes’ transposon-based STOP cloned in bacterial artificial chromosomes the ability to generate targeting vectors for and MGS kits “offer a convenient way to cre- (BACs), without the need using restriction conditional knockouts in mice, quickly and ate saturated libraries of mutated proteins in enzymes or ligases. The most popular systems efficiently. a single reaction with less hands-on time than are RecET, based on the recE and recT genes The main focus of Court’s lab is to under- any other method.” encoded by the cryptic RAC prophage that stand the molecular mechanisms of the Stratagene also strives for accuracy with a is present in some E. coli strains, and lambda recombination process, including the con- linear amplification method and high-fidelity Red, based on the homologous recombina- tributions of other phage-derived E. coli pro- DNA polymerase included in its QuikChange tion system of bacteriophage λ. The lambda teins, with a goal of optimizing the method mutagenesis kits, which can give results in a phage Red system is comprised of three for genetic engineering1: “We have demon- day. “Our QuikChange kits do not require functional parts: Redα (or Exo), a 5′-to-3′ strated that either dsDNA or ssDNA can be single-stranded DNA or labor-intensive, exonuclease; Redβ (or Bet or Beta), a protein used as the substrate for recombineering, and difficult-to-perform steps,” says Formosa. that binds to ssDNA overhangs generated by that the replication of the target is a required “Our trusted, proprietary non-PCR-based Redα; and Redγ (or Gam), an inhibitor of the component. We are aiming to understand QuikChange method ensures that only the major E. coli exonuclease and recombination what, if any, bacterial proteins are involved in parent strand is copied during cycling.” This complex, RecBCD. Red recombination.” line of products includes competent cells, but Several variations of the recombina- In the lab of Kenan Murphy, assistant pro- is also available in other versions depending tion system exist in which the three neces- fessor in molecular genetics and microbiolo- on researchers’ needs. “We even offer a ver- sary phage proteins Exo, Beta and Gam are gy at the University of Massachusetts Medical

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School, they are studying two aspects of although many additional utilities can be The lab of Don Court has also recently recombineering technology mechanisms2,3. imagined.” developed a set of plasmid tools to use for “First, we are examining the biochemistry of Work on the mycobacteria recombineer- recombineering in gram-negative bacteria. λ Bet and Exo proteins with dsDNA ends,” ing system continues in the Hatfull lab. The plasmids have been successfully used in says Murphy. “We have recently identified a In general, Hatfull says, it is versatile and Salmonella spp. and are likely to find applica- role for λ Exo in loading the ssDNA anneal- simple to use, but “one current limitation tions in other gram-negative bacteria. ing function Bet on to ssDNA. A specific is that the recovery of recombinants occurs complex of Bet and DNA ends is detected at somewhat lower frequencies than with Recombineering of mice, and flies, and on polyacrylamide gels that is not observed the E. coli systems. In and of itself this is men? when Bet is simply added to ssDNA after it is not a serious issue for most applications. Recombineering is already used to create generated by Exo.” However, the recombination frequencies mutants with relative ease, and there are are also dependent on the length of the more complex applications of this method Manipulating the bad bugs homologous DNA regions, and in practice on the horizon. Tim Townes, a profes- Another closely related application lurk- the number of recombinants recovered with sor in the Department of Biochemistry ing on the horizon is vaccine development. short sequences (that is, 50 bp) is very low. and Molecular Genetics at the University Murphy maintains that recombineering Thus we typically use ~500-bp segments of Alabama at Birmingham says, “we use could potentially “revolutionize the meth- of homology, which work well but require recombineering to create all of our con- ods of genetic manipulation in pathogenic somewhat complicated construction of tar- structs for production of knock-in mice. microorganisms, leading to faster identifi- geting substrates.” Before recombineering, complicated knock- cation of virulence genes, greater flexibil- ity in the genetic analysis of these genes and a Genetic engineering b Recombineering the speedy generation of bacterial mutants for vaccine development.” Another axis of Cassette PCR-amplified cassette research in Murphy’s lab is looking for Red or RecET-like systems in pathogenic bacte- Restiction ria, to develop recombineering for clinically + relevant species. Target in BAC Such an approach was recently taken by Julia van Kessel in the lab of Graham Hatfull, Multi-copy a professor in the Department of Biological drug R plasmid Sciences at the University of Pittsburg. Similar to the recombineering system in ori E. coli, Hatfull’s recombineering system was Ligation, developed specifically for mycobacteria4. transformation Transformation Plasmid/phage- “There were good reasons for thinking that a mediated homologous mycobacterial recombineering system would recombination be helpful, since in Mycobacterium tubercu- Cloned Recombinant cassette in BAC losis, constructing mutants by gene replace- drug R cassette ment is complicated by a relatively high level of illegitimate recombination,” says Hatfull. ori “We had heard anecdotally that colleagues + had attempted to use the E. coli systems in Target gene in BAC mycobacteria, but with little or no success.” Because the Hatfull lab studies myco- bacteriophages (phages that infect mycobacteria), they were able to find myco- bacteriophage genes that encode the recom- bination enzymes that would perform functions analogous to the phage-derived Classical homologous enzymes in E. coli. They identified the rare recombination steps Regions of homology recombination phage genes, Che9c genes 60 Target gene in BAC Recombinant cassette in BAC and 61; further characterization revealed that Restriction site the proteins that they encode were of the cor- Plasmid DNA rect functional type (that is, an exonuclease BAC DNA and a DNA pairing enzyme). “The primary PCR primers utility that we have described thus far is the Restriction enzyme use of mycobacterial recombineering for constructing knockout mutants in M. smeg- Classical recombinant DNA technology versus recombineering (reprinted from ref. 6 with permission matis and M. tuberculosis by allelic exchange, from Nature Reviews Genetics).

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or that you’re targeting a region of the chro- mosome that may be a cold spot for recom- bineering.” With these and perhaps other unknown pitfalls to navigate, some find it useful to rely on the dependability of a kit for already- tested reagents and procedures. So far, only one company, Gene Bridges, offers kits for researchers doing recombineering. The seven types of kits they offer include the construction of targeting vectors to create Takara Bio’s DNA polymerase and DNA Ligation kit including Mighty Mix. (Courtesy of Takara Bio.) transgenic mice (such as conditional knock- outs, knock-ins, single–base pair alterations); optimization of E. coli production strains; in plasmids required at least three months to Bellen thinks that tags will be impor- viral engineering, such as by making point construct. Now constructs can be produced, tant in the context of high-throughput mutations; and RNAi rescue analysis by BAC knock-in [embryonic stem] cells obtained, recombineering. “One important direction transgenesis, considered by many to be an and knock-in mice derived in 3 months.” will be high-throughput applications of important control for specificity in RNAi Likewise, in addition to making simple recombineering⎯for example, to tag each experiments. gene knockouts, Murphy’s lab uses recom- gene of a model organism in a genomic con- Based on the Red/recET recombination bineering for “BAC manipulation for mouse text and visualize its expression in vivo after system, each of the kits also contains con- genetics, chromosomal reporter constructs transgenesis. A variant of high-throughput trols to verify each step in the experiment. and genome reductions,” says Murphy. “The recombineering has been applied for the “Recombineering as a method is not difficult combination of site-specific recombinases modification of hundreds of BAC transgenes to apply,” says Harald Kranz, head of scien- (Flp and Cre) and counterselection markers in mice and is currently being used for the tific services at Gene Bridges. “Nevertheless, (sacB and rpsL) creates an almost limitless generation of thousands of gene-targeting we realized that it is very important to give ability to generate almost any type of chro- constructs.” customers enough control reactions so that mosomal alteration desired.” An eagerly anticipated application of they can set up the recombineering techno- Hugo Bellen, a Howard Hughes Medical recombineering is gene therapy in mam- logy step by step in their lab and adjust the Institute investigator at the Baylor College of malian cells. Court explains that the abil- reaction conditions to the equipment they Medicine, and colleagues are presently using ity to modify the chromosome and do ‘gene have. Giving the possibility to control the recombineering, in conjuction with other therapy’ in bacteria has progressed rapidly efficiency of every step during the reaction methods, to study Drosophila melanogaster in the last few years. His group’s studies with is very helpful.” genes5. They combined recombineering with the λ Red recombination functions have SeqWright now offers custom recom- conditionally amplifiable BAC that supports been critical for this advance. According to bineering services if you want to outsource two crucial features: first, gap-repair of large Court, efficient gene therapy in mammalian your experiment. Part of its custom subclon- DNA fragments using recombineering into cells using recombination based on the Red ing and mutagenesis services, SeqWright can a single copy plasmid, and second, plasmid functions is a real possibility. “Mammalian retrieve DNA fragments from large constructs copy induction to facilitate the isolation of viruses like HSV,” he explains, “use the same such as BACs, perform mutagenesis of large DNA for Drosophila transgenesis. Moreover, Red-like recombination functions as phage constructs and create gene-targeting vectors Bellen explains that “the single-copy feature λ and should be adaptable for the same pur- designed for genetically engineered mice. facilitates subsequent recombineering-medi- pose.” Whether you would like a simple cloning ated seamless mutagenesis, as shown by us, kit, or whether you are considering recom- and by Colin Dolphin and Ian Hope for Do try this at home bineering for gene therapy, the latest develop- Caernohabditis elegans.” Bellen’s group also But the use of recombineering is not limi- ments in cloning and mutagenesis will almost incorporated the fC31 system, previously ted to the few labs that have specialized in certainly help to speed your experiments shown to work in Drosophila in vivo to facili- its study and its applications to new systems. along accurately and efficiently. tate the integration of large DNA fragments. Murphy says that the challenges of using “There are virtually no limitations concern- recombineering as a method are certainly Caitlin Smith is a science writer based ing the method, since everything is homology not mechanical. “If you can perform a basic in Portland, Oregon, USA (caitlin. based,” says Bellen. PCR and punch a button on the electropora- [email protected]). Townes predicts that recombineering will tor, you can do recombineering,” he says. But be used to insert constructs encoding tandem that is not to say that experiments are always 1. Datta, S. et al. Gene 379, 109–115 (2006). 2. Murphy, K.C. & Campellone, K.G. BMC Mol. Biol. 4, affinity purification (TAP) tags into most, successful. “Sometimes we find recombineer- 11–22 (2003). if not all, endogenous transcription factor ing not working optimally, and it is usually 3. Campellone, K.G. et al. Mol. Microbiol. 63, 1468– 1481 (2007). genes in the mouse genome, so that tran- because our cells are not highly electrocom- 4. van Kessel, J.C., Hatfull, G.F. Nat. Methods. 4, scription factor complexes can be isolated petent, a drug marker is not expressed well 147–152 (2007). 5. Venken, K.J. et al. Science 314, 1747–1751 (2006). under physiological conditions and defined because of chromosomal context effects, the 6. Copeland, N.G., Jenkins, N.A. & Court D.L. Nat. Rev. by mass spectroscopy. drug concentration being used is too high, Genet. 2, 769–779 (2001).

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