Targeted mutagenesis by homologous recombination inD. melanogaster

Abstract

We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI 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. We recently described a method for targeted modification of the Drosophila genome through homologous recombination (HR). The ability to engineer specific changes into the genome is a highly useful adjunct to genetic investigation in any organism, but especially in a species with a completely determined genome sequence such as Drosophila melanogaster (Adams et al. 2000). This procedure had, until recently, been lacking in Drosophila. In our previous reports, we targeted two genes, rescuing a mutant allele of the first and generating a mutant allele of the second (Rong and Golic 2000, 2001). At this time there is a clear need for demonstrations of the generality of this technique. That is, can a variety of genes in different locations be modified by HR? There is also a need for the development of techniques that can produce mutant alleles of target genes. In this work, we address both issues by applying new methods for targeted mutagenesis of five autosomal genes.A variety of schemes has been produced for targeted gene modification in organisms such as yeast and mice (Rothstein 1991;Muller 1999). However, these methods rely critically on the ability to culture single cells and carry out selections for rare events. Because the targeting technique we use occurs in whole animals, we devised variant approaches for introducing mutations into chromosomal genes. The methods are mechanistically similar to those developed for yeast and mouse, but procedurally quite different, as they do not rely on chemical selections. Instead, at each step, arbitrary genetic markers with simple visible phenotypes are used for genetic screening. In our previous experiments, the frequency of targeted gene modification through HR varied from ∼ 1 in 500 gametes to ∼ 1 in 30,000 gametes. These frequencies are easily within reach of the power provided by genetic screening.To perform gene targeting in flies we use transgenic expression of FLP site-specific recombinase and I-SceI endonuclease to generate a targeting donor molecule in vivo. This donor molecule is derived from a third transgenic element: a P element carrying DNA homologous to the target locus. Within the P element, FLP Recombinase Target sites (FRTs) flank a segment of DNA from the target locus, and an I-SceI recognition site is placed within the target-homolog...

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation

“…We produced DDNp53 or Dp53 in p53-null flies. 35 First, we found that elevated levels of Dp53 or DDNp53 led to robust caspase activation, indicating that each isoform can induce apoptosis in the absence of the endogenous p53 gene (Figures 5a and b). Next, we observed that DDNp53 retained the ability to increase wg expression in the p53-null flies (Figures 5d and d 0 ).…”

“…The following transgenic and mutant fly stocks were used: MS1096-Gal4, en-Gal4, uas-lacZ and uas-RFP (Bloomington stock); uas-GFP, 43 rpr XRE -lacZ (rprZ) and dronc I29 (kind gifts from A Bergmann 25 ); p53R-GFPnls (p53 RE; a generous gift from J Abrams 15 ), rpr87, 44 deficiency (3L)H99 (Df(3L)H99, referred to as H99 45 ), PCNA-EmGFP 33 and p53-null (p53 [5A-1-4]). 35 The following genetic combinations were used to express transgenes in wing imaginal discs: (1) MS1096-Gal4,uas-GFP (MS10964GFP), (2) MS1096-Gal4;uas-Dp53 (MS10964Dp53), (3) MS1096-Gal4;uas-DDNp53 (MS10964 DDNp53), (4) enGal4/uas-Dp53 (en4Dp53), and (5) en-Gal4/uas-DDNp53 (en4DDNp53). Flies were raised under standard conditions at 25 1C.…”

Lessons from p53 in non-mammalian models

“…3,5 These findings were further supported by genetic lossof-function studies, which established that dmp53 is required for transcriptional induction of rpr proteins (rpr, hid, skl) and for radiation-induced apoptosis. 3 General development was not affected in the dmp53 mutants, [19][20][21] but mild defects in longevity and fertility were found. Where studied, dmp53 does not engage damage-induced cell cycle checkpoints and, consistent with this, the Drosophila ortholog of p21 is unresponsive to irradiation.…”

Xenopus, the next generation: X. Tropicalis genetics and genomics

“…Another strategy that may be promising for X. tropicalis is the homologous recombination strategy developed by Golic and collaborators for Drosophila Golic, 2000, 2001;Rong et al, 2002). In this approach, in vivo excision of an introduced targeting construct by using a rare-cutting restriction enzyme enhances homologous integration by presentation of recombinogenic DNA ends.…”