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Applications of Xâ Downloaded from genesdev.cshlp.org on June 30, 2009 - Published by Cold Spring Harbor Laboratory Press ‘IN n e-fr £ Development Targeted mutagenesis by homologous recombination in D. melanogaster Yikang S. Rong, Simon W. Titen, Heng B. Xie, et al. G e n e s D e v . 2002 16: 1568-1581 Access the most recent version at doi:10.1101/gad.986602 References This article cites 43 articles, 24 of which can be accessed free at: http://genesdev.cshlp.org/content/16/12/1568.fuN.html#ref-Nst-1 Article cited in: http://genesdev.cshlp.org/content/16/12/1568.full.html#related-urls Email alerting Receive free email alerts when new articles cite this article - sign up in the box at the service top right corner of the article or click here To subscribe to Genes & Development go to: http://genesdev.cshlp.org/subscriptions Cold Spring Harbor Laboratory Press Downloaded from genesdev.cshlp.org on June 30, 2009 - Published by Cold Spring Harbor Laboratory Press m utagenesis by recom bination in D . m elanogaster Yikang S. Rong,1,2,4 Simon W. Titen,1,2 Heng B. Xie,1,2 Mary M. Golic,1,2 Michael Bastiani,1 Pradip Bandyopadhyay,1 Baldomero M. Olivera,1 Michael Brodsky,3,5 Gerald M. Rubin,3 and Kent G. Golic1,2,6 departm ent of Biology, University of Utah, Salt Lake City, Utah 84112, USA; 2Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA; 3Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA Wc used a recently developed method to produce mutant alleles of five endogenous D r o s o p h ila genes, including the homolog of the p 5 3 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-Scel endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced. [Key Words: Gene targeting; Drosophila: recombination; FLP; I-Scel] Received February 21, 2002; revised version accepted May 3, 2002. We recently described a method for targeted modifica­ technique wc use occurs in whole animals, wc devised tion of the D rosoph ila genome through homologous re­ variant approaches for introducing mutations into chro­ combination (HR). The ability to engineer specific mosomal genes. The methods arc mechanistically simi­ changes into the genome is a highly useful adjunct to lar to those developed for yeast and mouse, but procc- genetic investigation in any organism, but especially in a durally quite different, as they do not rely on chemical species with a completely determined genome sequence selections. Instead, at each step, arbitrary genetic mark­ such as Drosophila melanogaster (Adams ct al. 2000). ers with simple visible phenotypes arc used for genetic This procedure had, until recently, been lacking in D ro ­ screening. In our previous experiments, the frequency of soph ila. In our previous reports, wc targeted two genes, targeted gene m odification through H R varied from ~1 in rescuing a mutant allele of the first and generating a 500 gametes to ~1 in 30,000 gametes. These frequencies m utant allele of the second (Rong and Golic 2000, 2001). arc easily within reach of the power provided by genetic At this time there is a clear need for demonstrations of screening. the generality of this technique. That is, can a variety of To perform gene targeting in flics wc use transgenic genes in different locations be modified by HR? There is expression of FLP site-specific recombinase and I-Scel also a need for the development of techniques that can endonuclease to generate a targeting donor molecule in produce mutant alleles of target genes. In this work, wc vivo. This donor molecule is derived from a third trans­ address both issues by applying new methods for tar­ genic clement: a P clement carrying DNA homologous geted mutagenesis of five autosomal genes. to the target locus. Within the P clement, FLP Rccom- A variety of schemes has been produced for targeted binasc Target sites {FRTs) flank a segment of DNA from gene modification in organisms such as yeast and mice the target locus, and an I-Scel recognition site is placed (Rothstcin 1991; Muller 1999). However, these methods within the target-homologous sequence. The expression rely critically on the ability to culture single cells and of FLP and I-Scel excises the donor sequence from its carry out selections for rare events. Because the targeting initially random chromosomal location and generates a double-strand break (DSB) within the sequence that is homologous to the target gene. The DSB stimulates HR of the donor and the corresponding chromosomal target Present addresses: ’Laboratory of Molecular Cell Biology, National Can­ locus (Fig. 1). Actual targeting events may be recovered cer Institute, National Institutes of Health, Bethesda, MD 20892, USA; by screening for movement of an eye-color marker gene ^Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01655, USA. that is carried into the target locus as part of the donor “Corresponding author. DNA. An alternative, more rapid, screening method has E-MAIL [email protected]; FAX (816) 926-2065. Articlc and publication arc at http://www.gcncsdcv.org/cgi/doi/10.1101/ also been described (Rong and Golic 2001). The process gad.986602. of gene targeting in D rosoph ila is diagramed in Figure 2. 1568 GENES & DEVELOPMENT 16:1568-1581 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 S5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on June 30, 2009 - Published by Cold Spring Harbor Laboratory Press Drosophila gene targeting Figure 1. General form of ends-in gene tar­ geting. The recombinogenic donor molecule is generated by FLP and l-Scel action on a P- element donor construct, causing excision of the target-homologous sequence and the marker gene, and cutting at the l-Scel site. HR with the chromosomal target locus generates a tandem duplication of the sequence that was present in the donor with incorporation of the marker gene between duplicated target segments. Mutations engineered into the do­ nor (indicated as asterisks) may be carried into both copies of the target gene. In this paper we apply two new procedures to generate These results extend that observation to show that a m utant alleles of genes that are identified only by DNA significantly enhanced efficiency of targeting in females sequence. Knowledge of the mutant phenotype is not also applies to autosomal target genes. We conclude that required or relied on in these procedures. Moreover, the relatively inefficient targeting in males is not related to end product of one protocol is a precise substitution of an lack of a homolog because this difference occurs for both engineered mutant allele for the wild-type allele: no ex­ autosomal and X-linked genes. ogenous DNA is left behind at the target locus other Another significant difference between males and fe­ than the introduced mutation. males is that the targeted (homologous) recombinants We used these procedures to specifically mutate sev­ outnumber the nontargeted (illegitimate) events by 3:1 eral endogenous genes at a variety of chromosomal loci. in females, but in males that relationship is reversed, We show that the process is sufficiently efficient and with nontargeted events outnumbering targeted recom­ flexible to be generally useful for modification of the binants by alm ost 2:1 (P = 0.005). D rosoph ila genom e. Because of the increased efficiency of targeting in fe­ males, targeting of N L a z (N eu ral L azarillo; Sanchez et al. 2000), GC (y-glutamyl carboxylase ; Li et al. 2000; R esults Walker et al. 2001), and C G 11305 (a homolog of the yeast Sir2 gene; Gasser and Cockell 2001) was done only General parameters of gene targeting in Drosophila through females. For the genes targeted in this work, We constructed a P-element vector, pTV2, to be used as independent homologous recombination events were re­ a general transformation vector for gene targeting. This covered from females at an average (unweighted) rate of vector carries an FRT-flanked w+ gene jw*sj that is used ~1 per 40 vials. These crosses typically produce -100 for transformation, and to track movement of the donor progeny per vial. Targeting events were often recovered to detect gene targeting. We used transformants of donor in clusters (but scored as single events), making the per constructs in TV2 to carry out targeted mutagenesis of gamete rate of targeting better than 1 in 4000. Because five genes (Fig. 3). Only one of these genes was known by there are at least two and sometimes four (in G2) target existing mutants prior to this work. Table 1 shows the chromosomes per cell, the cellular targeting rate is at efficiency of targeting obtained for each gene. Two gen­ least 1 in 2000. eral trends appear. First, targeting is substantially more Substantial variation in efficiency occurred at different efficient in the female germ line than in the male germ target genes. At C G 11305, targeting averaged 1 event per line. Looking at the data for pug {pugilist; Rong and Golic 17 vials, but at GC, it was as infrequent as 1 event per 1998) and p 5 3 (encoding the D rosoph ila homolog of the 340 vials {P = 0.0003).
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