- Email: [email protected]
Gene-targeting in Drosophila validated
Research Update
TRENDS in Genetics Vol.17 No.10 October 2001
549
Research News
Gene-targeting in Drosophila validated Gregory B. Gloor Until recently, the use of reverse genetics by gene targeting, the method used to great effect in Saccharomyces cerevisiae and mouse, was glaringly absent in Drosophila. This deficiency no longer exists because of recent publications by Rong and Golic outlining successful gene targeting in Drosophila melanogaster. Their most recent report answers several doubts about the general feasibility of gene targeting in Drosophila. However, the very low targeting frequency at the locus raises new concerns about the general utility of the method.
Drosophila is an attractive research organism partly because of the wealth of sophisticated and ingenious genetic tools available to modify the genome (e.g. Refs 1–3). In addition, the recent successful application of dsRNA-mediated interference (RNAi) to generate a hypomorphic phenocopy (i.e. a phenotype not caused by genetic mutation) in adult flies4 means that there could be rapid alternative means to examine mutant phenotypes. However, this RNAi experiment failed to replicate a true null allele, showing the necessity of knockout and mutagenesis experiments. Many of these tools address, in one way or another, the lack of a gene-knockout system in Drosophila that is analogous to those used in other organisms. In Drosophila, unlike in mammals, transplanted cultured cells cannot form part of the germ line5. This limitation, more than any other, has prevented the development of a geneknockout system in Drosophila. Gene targeting requires cultured cells because a linear gene-targeting vector must be introduced into the cells. The host-cell recombination machinery then assembles on the ends of the introduced DNA, producing rare integration events into the appropriate target site. As shown in Fig. 1a, Rong and Golic6 realized that they could produce a linear DNA molecule appropriate for gene targeting in vivo by circularizing an integrated construct with FLP recombinase, and subsequently digesting it with the I-SceI endonuclease. Several other groups had tried similar experiments with limited success in Drosophila and in yeast7–9. http://tig.trends.com
Concerns with the initial experiment
In their initial experiment early last year, Rong and Golic6 demonstrated gene targeting in Drosophila by reverting a mutation in the X-linked yellow gene. The generality of their method was questioned10, in part because Bellaiche et al.9 had recently published a similar procedure in which they had failed to recover gene targeting events at a different locus, even though Bellaiche et al. had examined 100 times more flies than Rong and Golic. Engels proposed several reasons for this discrepancy10. He suggested that the high rate of targeting observed in the female but not male germ line, and the variety of products recovered might be due to the position of the yellow gene close to the telomere. The proximity of the yellow gene to the telomere made it possible that break-induced replication (BIR), which is similar to, but distinct from, homologous recombination11,12, was responsible for at least some of the gene targeting events that were observed at the yellow gene. Furthermore, the restriction of Rong and Golic’s method to ends-in targeting further suggested that functional null alleles might be difficult to generate by this method because ends-in targeting produces a duplication of the target site locus (Fig. 1a). These features conspired to suggest that reverse genetics in Drosophila might be restricted to endsin gene targeting by BIR at those genes that were located close to a telomere. Targeting a mutation to an arbitrary locus
Rong and Golic’s13 second application of reverse genetics to the Drosophila genome addresses some of these issues head on. For this experiment, they chose to target the pugilist (pug) locus, which encodes methylenetetrahydrofolate dehydrogenase, a non-essential gene with a ‘subtle eye’ phenotype. The pug gene was well chosen for several reasons. First, this gene is located in the middle of the right arm of chromosome 3, where it is more than 20 Mb distant from the nearest telomere. Therefore, break-induced recombination is an unlikely mechanism for gene targeting. Second, they used only
2.5 kb of pug locus DNA in their targeting vector, as opposed to 8 kb in their original experiment7. This means that only a small fragment of a typical gene could be required for gene targeting, simplifying vector construction (but see discussion below). Third, they recovered a functional null allele of pug by including one point mutation in the pug gene on each side of the I-SceI site. Because ends-in gene targeting always generates a duplication of the targeted gene, they recovered two different point mutations, one into each of the duplicated pug genes. Thus, they demonstrated the general feasibility of this method, even for genes that present a small target for recombination. There are two other noteworthy features reported in Rong and Golic’s second paper13. First, the frequency of gene targeting at the pug locus was less than one event per 25 000 gametes. This is much lower than the frequency observed at the yellow locus, which was about one (correctly) targeted event per 1500 gametes6. In their first report, Rong and Golic also found a large difference in the gene targeting frequency between the male and female germ lines, with targeting occurring at least an order of magnitude more frequently in the female germ line6. In this report, Rong and Golic13 recovered two targeted pug insertions from the female germ line (about 50 000 gametes) and none from the male germ line (about 34 000 gametes). Although their second experiment failed to resolve conclusively the question of whether targeting could occur in either male or female germ line at an autosomal site, prudence suggests collecting events from the female germ line. The frequency of gene targeting in the female germ line at the pugilist locus suggests that Bellaiche et al.9 might have been able to recover a targeted insertion at the white locus if they had conducted their experiments in the female germ line rather than in the male germ line. Second, Rong and Golic13 modified their original procedure to reduce the high background of non-excised vector sequences. As shown in Fig. 1, excision of
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02419-2
Research Update
550
(a) Ends-in targeting
TRENDS in Genetics Vol.17 No.10 October 2001
(b) Ends-out targeting
I-Sce I cut site FRT
I-Sce I cut site
white gene FRT
FRT
white gene FRT
Step 1: Excise Express FLP recombinase
Step 2: Linearize Express I-Sce I endonuclease
Step 3: Integrate Host recombination
Duplication of target locus
Replacement of target locus TRENDS in Genetics
Fig. 1. An outline of the gene targeting method in Drosophila. The ends-in gene targeting method used by Rong and Golic13 (a), and an ends-out gene targeting that could be used to generate gene replacements (b). In either case, the targeting vector is first integrated into the Drosophila genome by P-element-mediated germ-line transformation21. The vector contains a white gene (blue), which serves as the visible marker for transformation and, eventually, for gene targeting. An I-SceI endonuclease recognition site (scissors) bisects the sequence (purple) that is homologous to the target locus (green). In an end-in targeting vector the sequence homologous to the target locus and the target locus are co-linear. In an ends-out targeting vector one end of this sequence is inverted. In both situations, two FLP recombinase recognition sites in direct orientation (FRT, yellow) flank the entire targeting construct. Expression of the FLP recombinase, which was taken from Saccharomyces cerevisiae and adapted for use in Drosophila22, causes site-specific recombination between the paired FRT sites resulting in the release of the targeting vector from the genome as a DNA circle. Digestion of this circle by the I-SceI endonuclease linearizes it, releasing the targeting vector. Recombination between the linear vector and the target locus inserts the targeting vector into the host genome. This results in a duplication of the locus with ends-in targeting or replacement of the locus with ends-out targeting. Correctly targeted insertions are identified by linkage analysis if the targeting vector is located on a chromosome different from the one that targeting is directed towards.
the targeting vector from the chromosome is required. This excision is not completely efficient; in our hands about 0.1–1% of the vector sequences do not excise, and lead to white+ progeny that are indistinguishable from true targeting events until they are mapped. In my experience, these false positives, although easy to detect by examining their segregation patterns, contribute unduly to the amount of work involved in recovering a targeting event. Rong and Golic13 suggest that all female flies, in which the targeting is occurring, be mated to flies homozygous for a P{hsp FLP} transgene. The resulting progeny of this mating should be subjected to a heat shock to induce expression of the FLP recombinase, thus causing recombination between paired FLP recombinase recognition (FRT) sites, as shown in Fig. 1. Those progeny that carry a non-excised targeting vector will have a http://tig.trends.com
white or mosaic eye color because of this treatment. True gene targeting events can be distinguished because they will have a solid eye color because the white gene in these events is not flanked by FRT elements. Is targeting the method of choice for mutagenesis in Drosophila?
These results raise a question: is gene targeting going to be the method choice for mutagenesis in Drosophila? I believe that there are several reasons that the technology is not yet mature enough for the majority of Drosophila labs to adopt, but that it will rapidly improve in the near future. This method needs to be extended and improved in two main ways. First, the large number of flies that were scored to recover a mutant allele of the pug locus suggests that careful attention to vector design will probably
prove as important in Drosophila as it has in the mouse. For example, in my lab, we were unsuccessful in recovering a genetargeting event to a locus located near the middle of the second chromosome after scoring more than 30 000 progeny derived from the female germ line. In our case, we had 2 kb of flanking homology (500 bp on one side and 1.5 kb on the other). This was similar to the amount used at the pug locus. It is likely that we failed to recover a gene targeting event because more flanking homology might be required by this method than the small amount required for P-element-induced gene replacement14. For example, the frequency of targeted gene insertion in mouse ES cells increases exponentially with increasing amounts of sequence homology and saturates when there is about 14 kb of homology between the vector and target locus15. A further increase in the frequency of gene targeting is seen when the vector sequence and the target locus are isogenic16. Without these optimizations, it seems probable that the number of flies that need to be scored for gene targeting will generally approach the number that is required by more traditional mutagenesis schemes in Drosophila. Second, the ends-in targeting method causes a duplication of the targeted locus. This makes it hard to know whether a true null allele is generated, and furthermore makes the targeting of other allelic variants difficult. A means of conducting ends-out gene targeting in Drosophila (shown in Fig. 1b), that is formally similar to that used in the yeast whole-genome knockout project17, would certainly be very useful to complement the information generated by the Drosophila genome project18. The ends-out method generates complete deletions of the target locus and, in principle, can make any arbitrary change in the genome19. Although endsout targeting is likely to be less efficient than ends-in targeting19, one way to achieve it would be to conduct both a positive screen for gene targeting by a chemical selection and a negative selection for the chromosome carrying the targeting vector. Methods that follow this philosophy have been used to great effect in mouse ES cells20. We are currently testing this idea by incorporating the targeting vector on a chromosome carrying a temperature sensitive mutation, and by including a selectable gene that can co-target with the white
Research Update
gene to the target locus. We expect that such a configuration would permit the rapid scoring of hundreds of thousands of progeny, and thus add ends-out targeting to an arbitrary locus in Drosophila to the Drosophilist’s armory. Acknowledgements
Work in my lab is supported by a grant from the Canadian Institutes of Health Research. I am grateful to Dr Kent Golic for supplying information before publication, and to two anonymous reviewers for their helpful comments. References 1 Engels, W.R. (1997) Invasions of P elements. Genetics 145, 11–15 2 Golic, M.M. et al. (1997) FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes. Nucleic Acids Res. 25, 3665–3671 3 Golic, K.G. and Golic, M.M. (1996) Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144, 1693–1711 4 Martinek, S. and Young, M.W. (2000) Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats. Genetics 156, 1717–1725 5 Ashburner, M. (1989) Drosophila, A Laboratory Handbook, Cold Spring Harbor Press
TRENDS in Genetics Vol.17 No.10 October 2001
6 Rong, Y.S. and Golic, K.G. (2000) Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018 7 Negritto, M.T. et al. (1997) Influence of DNA sequence identity on efficiency of targeted gene replacement. Mol. Cell. Biol. 17, 278–286 8 Leung, W. et al. (1997) Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction. Proc. Natl. Acad. Sci. U. S. A. 94, 6851–6856 9 Bellaiche, Y. et al. (1999) I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics 152, 1037–1044 10 Engels, W.R. (2000) Reversal of fortune for Drosophila geneticists? Science 288, 1973–1975 11 Kuzminov, A. (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63, 751–813 12 Haber, J.E. (2000) Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264 13 Rong, Y. and Golic, K. (2001) A targeted gene knockout in Drosophila. Genetics 157, 1307–1312 14 Dray, T. and Gloor, G.B. (1997) Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster. Genetics 147, 689–699 15 Deng, C. and Capecchi, M.R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12, 3365–3371
551
16 te Riele, H. et al. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U. S. A. 89, 5128–5132 17 Winzeler, E.A. et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 18 Adams, M.D. et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 19 Hastings, P.J. et al. (1993) Ends-in vs. ends-out recombination in yeast. Genetics 135, 973–980 20 Mansour, S.L. et al. (1988) Disruption of the protooncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 21 Spradling, A.C. and Rubin, G.M. (1982) Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341–347 22 Golic, K.G. and Lindquist, S. (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499–509
Gregory B. Gloor Dept of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1. e-mail: [email protected]
Causes of the phenotype–genotype dissociation in DiGeorge syndrome: clues from mouse models Annalisa Botta, Francesca Amati and Giuseppe Novelli Heterozygous deletions within human chromosome 22q11.2 are the genetic basis of DiGeorge syndrome (DGS), the most common deletion syndrome known in humans. To elucidate the molecular mechanism underlying this disease, researchers focused their attention on mouse genetics, creating animals carrying deletions of regions syntenic to the human DGS locus or targeted mutations of individual genes. Although some of these mouse mutants recapitulate many of the phenotypic features of DGS, they do not fully explain the complex genetics of the human disease. This article gives a short overview and comments on the most recent advances in this field.
DiGeorge syndrome (DGS; OMIM 188400) is a developmental disorder, characterized by a wide spectrum of clinical symptoms including conotruncal cardiac anomalies and facial, thymic, thyroid and http://tig.trends.com
parathyroid defects. Additional phenotypic features are psychiatric disorders, ocular defects, upper-limb malformations, renal and urological tract malformations, cerebellar atrophy, tracheal defects and hearing loss1. These different abnormalities are believed to have a common etiology, based on defective differentiation of neural crest cells2. More than 90% of DGS patients have detectable deletions within a 3-Mb region (TDR, typically deleted region) of chromosome 22q11.2 (Ref. 3). On the basis of comparative deletion mapping data from rare patients with atypical deletions and a clinically similar phenotype, the TDR has been subdivided into five critical non-overlapping intervals (interval 1–5) in which at least 40 genes have been located4 (Fig. 1). The absence of a genotype–phenotype correlation in these cases and the lack of mutation in several TDR genes from patients with no
detectable 22q11.2 deletion5–11, mean that it has not been possible to identify single genes that cause the disorder. To understand the molecular basis of this disease, researchers focused their attention on animal models by creating mice with hemizygous deletions in regions of chromosome 16 that correspond to the human DGS locus12,13. In 1999, Lindsay et al.14, created the first animal model of DGS using Cre-loxP chromosome engineering in mice. They deleted a 1.2-Mb fragment (named Df1) spanning a segment of the murine chromosome 16B that is syntenic to human 22q11.2 and that contains at least 20 orthologs of genes commonly deleted in DGS/VCFS patients (Fig. 1). About 10% of mice heterozygous for this chromosomal deletion (Df1/+) showed neonatal mortality; the rest were viable and fertile. Depending on the age of examination, 18–26% of Df1/+ mice showed defective development of the
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02438-6
TRENDS in Genetics Vol.17 No.10 October 2001
549
Research News
Gene-targeting in Drosophila validated Gregory B. Gloor Until recently, the use of reverse genetics by gene targeting, the method used to great effect in Saccharomyces cerevisiae and mouse, was glaringly absent in Drosophila. This deficiency no longer exists because of recent publications by Rong and Golic outlining successful gene targeting in Drosophila melanogaster. Their most recent report answers several doubts about the general feasibility of gene targeting in Drosophila. However, the very low targeting frequency at the locus raises new concerns about the general utility of the method.
Drosophila is an attractive research organism partly because of the wealth of sophisticated and ingenious genetic tools available to modify the genome (e.g. Refs 1–3). In addition, the recent successful application of dsRNA-mediated interference (RNAi) to generate a hypomorphic phenocopy (i.e. a phenotype not caused by genetic mutation) in adult flies4 means that there could be rapid alternative means to examine mutant phenotypes. However, this RNAi experiment failed to replicate a true null allele, showing the necessity of knockout and mutagenesis experiments. Many of these tools address, in one way or another, the lack of a gene-knockout system in Drosophila that is analogous to those used in other organisms. In Drosophila, unlike in mammals, transplanted cultured cells cannot form part of the germ line5. This limitation, more than any other, has prevented the development of a geneknockout system in Drosophila. Gene targeting requires cultured cells because a linear gene-targeting vector must be introduced into the cells. The host-cell recombination machinery then assembles on the ends of the introduced DNA, producing rare integration events into the appropriate target site. As shown in Fig. 1a, Rong and Golic6 realized that they could produce a linear DNA molecule appropriate for gene targeting in vivo by circularizing an integrated construct with FLP recombinase, and subsequently digesting it with the I-SceI endonuclease. Several other groups had tried similar experiments with limited success in Drosophila and in yeast7–9. http://tig.trends.com
Concerns with the initial experiment
In their initial experiment early last year, Rong and Golic6 demonstrated gene targeting in Drosophila by reverting a mutation in the X-linked yellow gene. The generality of their method was questioned10, in part because Bellaiche et al.9 had recently published a similar procedure in which they had failed to recover gene targeting events at a different locus, even though Bellaiche et al. had examined 100 times more flies than Rong and Golic. Engels proposed several reasons for this discrepancy10. He suggested that the high rate of targeting observed in the female but not male germ line, and the variety of products recovered might be due to the position of the yellow gene close to the telomere. The proximity of the yellow gene to the telomere made it possible that break-induced replication (BIR), which is similar to, but distinct from, homologous recombination11,12, was responsible for at least some of the gene targeting events that were observed at the yellow gene. Furthermore, the restriction of Rong and Golic’s method to ends-in targeting further suggested that functional null alleles might be difficult to generate by this method because ends-in targeting produces a duplication of the target site locus (Fig. 1a). These features conspired to suggest that reverse genetics in Drosophila might be restricted to endsin gene targeting by BIR at those genes that were located close to a telomere. Targeting a mutation to an arbitrary locus
Rong and Golic’s13 second application of reverse genetics to the Drosophila genome addresses some of these issues head on. For this experiment, they chose to target the pugilist (pug) locus, which encodes methylenetetrahydrofolate dehydrogenase, a non-essential gene with a ‘subtle eye’ phenotype. The pug gene was well chosen for several reasons. First, this gene is located in the middle of the right arm of chromosome 3, where it is more than 20 Mb distant from the nearest telomere. Therefore, break-induced recombination is an unlikely mechanism for gene targeting. Second, they used only
2.5 kb of pug locus DNA in their targeting vector, as opposed to 8 kb in their original experiment7. This means that only a small fragment of a typical gene could be required for gene targeting, simplifying vector construction (but see discussion below). Third, they recovered a functional null allele of pug by including one point mutation in the pug gene on each side of the I-SceI site. Because ends-in gene targeting always generates a duplication of the targeted gene, they recovered two different point mutations, one into each of the duplicated pug genes. Thus, they demonstrated the general feasibility of this method, even for genes that present a small target for recombination. There are two other noteworthy features reported in Rong and Golic’s second paper13. First, the frequency of gene targeting at the pug locus was less than one event per 25 000 gametes. This is much lower than the frequency observed at the yellow locus, which was about one (correctly) targeted event per 1500 gametes6. In their first report, Rong and Golic also found a large difference in the gene targeting frequency between the male and female germ lines, with targeting occurring at least an order of magnitude more frequently in the female germ line6. In this report, Rong and Golic13 recovered two targeted pug insertions from the female germ line (about 50 000 gametes) and none from the male germ line (about 34 000 gametes). Although their second experiment failed to resolve conclusively the question of whether targeting could occur in either male or female germ line at an autosomal site, prudence suggests collecting events from the female germ line. The frequency of gene targeting in the female germ line at the pugilist locus suggests that Bellaiche et al.9 might have been able to recover a targeted insertion at the white locus if they had conducted their experiments in the female germ line rather than in the male germ line. Second, Rong and Golic13 modified their original procedure to reduce the high background of non-excised vector sequences. As shown in Fig. 1, excision of
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02419-2
Research Update
550
(a) Ends-in targeting
TRENDS in Genetics Vol.17 No.10 October 2001
(b) Ends-out targeting
I-Sce I cut site FRT
I-Sce I cut site
white gene FRT
FRT
white gene FRT
Step 1: Excise Express FLP recombinase
Step 2: Linearize Express I-Sce I endonuclease
Step 3: Integrate Host recombination
Duplication of target locus
Replacement of target locus TRENDS in Genetics
Fig. 1. An outline of the gene targeting method in Drosophila. The ends-in gene targeting method used by Rong and Golic13 (a), and an ends-out gene targeting that could be used to generate gene replacements (b). In either case, the targeting vector is first integrated into the Drosophila genome by P-element-mediated germ-line transformation21. The vector contains a white gene (blue), which serves as the visible marker for transformation and, eventually, for gene targeting. An I-SceI endonuclease recognition site (scissors) bisects the sequence (purple) that is homologous to the target locus (green). In an end-in targeting vector the sequence homologous to the target locus and the target locus are co-linear. In an ends-out targeting vector one end of this sequence is inverted. In both situations, two FLP recombinase recognition sites in direct orientation (FRT, yellow) flank the entire targeting construct. Expression of the FLP recombinase, which was taken from Saccharomyces cerevisiae and adapted for use in Drosophila22, causes site-specific recombination between the paired FRT sites resulting in the release of the targeting vector from the genome as a DNA circle. Digestion of this circle by the I-SceI endonuclease linearizes it, releasing the targeting vector. Recombination between the linear vector and the target locus inserts the targeting vector into the host genome. This results in a duplication of the locus with ends-in targeting or replacement of the locus with ends-out targeting. Correctly targeted insertions are identified by linkage analysis if the targeting vector is located on a chromosome different from the one that targeting is directed towards.
the targeting vector from the chromosome is required. This excision is not completely efficient; in our hands about 0.1–1% of the vector sequences do not excise, and lead to white+ progeny that are indistinguishable from true targeting events until they are mapped. In my experience, these false positives, although easy to detect by examining their segregation patterns, contribute unduly to the amount of work involved in recovering a targeting event. Rong and Golic13 suggest that all female flies, in which the targeting is occurring, be mated to flies homozygous for a P{hsp FLP} transgene. The resulting progeny of this mating should be subjected to a heat shock to induce expression of the FLP recombinase, thus causing recombination between paired FLP recombinase recognition (FRT) sites, as shown in Fig. 1. Those progeny that carry a non-excised targeting vector will have a http://tig.trends.com
white or mosaic eye color because of this treatment. True gene targeting events can be distinguished because they will have a solid eye color because the white gene in these events is not flanked by FRT elements. Is targeting the method of choice for mutagenesis in Drosophila?
These results raise a question: is gene targeting going to be the method choice for mutagenesis in Drosophila? I believe that there are several reasons that the technology is not yet mature enough for the majority of Drosophila labs to adopt, but that it will rapidly improve in the near future. This method needs to be extended and improved in two main ways. First, the large number of flies that were scored to recover a mutant allele of the pug locus suggests that careful attention to vector design will probably
prove as important in Drosophila as it has in the mouse. For example, in my lab, we were unsuccessful in recovering a genetargeting event to a locus located near the middle of the second chromosome after scoring more than 30 000 progeny derived from the female germ line. In our case, we had 2 kb of flanking homology (500 bp on one side and 1.5 kb on the other). This was similar to the amount used at the pug locus. It is likely that we failed to recover a gene targeting event because more flanking homology might be required by this method than the small amount required for P-element-induced gene replacement14. For example, the frequency of targeted gene insertion in mouse ES cells increases exponentially with increasing amounts of sequence homology and saturates when there is about 14 kb of homology between the vector and target locus15. A further increase in the frequency of gene targeting is seen when the vector sequence and the target locus are isogenic16. Without these optimizations, it seems probable that the number of flies that need to be scored for gene targeting will generally approach the number that is required by more traditional mutagenesis schemes in Drosophila. Second, the ends-in targeting method causes a duplication of the targeted locus. This makes it hard to know whether a true null allele is generated, and furthermore makes the targeting of other allelic variants difficult. A means of conducting ends-out gene targeting in Drosophila (shown in Fig. 1b), that is formally similar to that used in the yeast whole-genome knockout project17, would certainly be very useful to complement the information generated by the Drosophila genome project18. The ends-out method generates complete deletions of the target locus and, in principle, can make any arbitrary change in the genome19. Although endsout targeting is likely to be less efficient than ends-in targeting19, one way to achieve it would be to conduct both a positive screen for gene targeting by a chemical selection and a negative selection for the chromosome carrying the targeting vector. Methods that follow this philosophy have been used to great effect in mouse ES cells20. We are currently testing this idea by incorporating the targeting vector on a chromosome carrying a temperature sensitive mutation, and by including a selectable gene that can co-target with the white
Research Update
gene to the target locus. We expect that such a configuration would permit the rapid scoring of hundreds of thousands of progeny, and thus add ends-out targeting to an arbitrary locus in Drosophila to the Drosophilist’s armory. Acknowledgements
Work in my lab is supported by a grant from the Canadian Institutes of Health Research. I am grateful to Dr Kent Golic for supplying information before publication, and to two anonymous reviewers for their helpful comments. References 1 Engels, W.R. (1997) Invasions of P elements. Genetics 145, 11–15 2 Golic, M.M. et al. (1997) FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes. Nucleic Acids Res. 25, 3665–3671 3 Golic, K.G. and Golic, M.M. (1996) Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144, 1693–1711 4 Martinek, S. and Young, M.W. (2000) Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats. Genetics 156, 1717–1725 5 Ashburner, M. (1989) Drosophila, A Laboratory Handbook, Cold Spring Harbor Press
TRENDS in Genetics Vol.17 No.10 October 2001
6 Rong, Y.S. and Golic, K.G. (2000) Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018 7 Negritto, M.T. et al. (1997) Influence of DNA sequence identity on efficiency of targeted gene replacement. Mol. Cell. Biol. 17, 278–286 8 Leung, W. et al. (1997) Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction. Proc. Natl. Acad. Sci. U. S. A. 94, 6851–6856 9 Bellaiche, Y. et al. (1999) I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics 152, 1037–1044 10 Engels, W.R. (2000) Reversal of fortune for Drosophila geneticists? Science 288, 1973–1975 11 Kuzminov, A. (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63, 751–813 12 Haber, J.E. (2000) Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264 13 Rong, Y. and Golic, K. (2001) A targeted gene knockout in Drosophila. Genetics 157, 1307–1312 14 Dray, T. and Gloor, G.B. (1997) Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster. Genetics 147, 689–699 15 Deng, C. and Capecchi, M.R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12, 3365–3371
551
16 te Riele, H. et al. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U. S. A. 89, 5128–5132 17 Winzeler, E.A. et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 18 Adams, M.D. et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 19 Hastings, P.J. et al. (1993) Ends-in vs. ends-out recombination in yeast. Genetics 135, 973–980 20 Mansour, S.L. et al. (1988) Disruption of the protooncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 21 Spradling, A.C. and Rubin, G.M. (1982) Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341–347 22 Golic, K.G. and Lindquist, S. (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499–509
Gregory B. Gloor Dept of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1. e-mail: [email protected]
Causes of the phenotype–genotype dissociation in DiGeorge syndrome: clues from mouse models Annalisa Botta, Francesca Amati and Giuseppe Novelli Heterozygous deletions within human chromosome 22q11.2 are the genetic basis of DiGeorge syndrome (DGS), the most common deletion syndrome known in humans. To elucidate the molecular mechanism underlying this disease, researchers focused their attention on mouse genetics, creating animals carrying deletions of regions syntenic to the human DGS locus or targeted mutations of individual genes. Although some of these mouse mutants recapitulate many of the phenotypic features of DGS, they do not fully explain the complex genetics of the human disease. This article gives a short overview and comments on the most recent advances in this field.
DiGeorge syndrome (DGS; OMIM 188400) is a developmental disorder, characterized by a wide spectrum of clinical symptoms including conotruncal cardiac anomalies and facial, thymic, thyroid and http://tig.trends.com
parathyroid defects. Additional phenotypic features are psychiatric disorders, ocular defects, upper-limb malformations, renal and urological tract malformations, cerebellar atrophy, tracheal defects and hearing loss1. These different abnormalities are believed to have a common etiology, based on defective differentiation of neural crest cells2. More than 90% of DGS patients have detectable deletions within a 3-Mb region (TDR, typically deleted region) of chromosome 22q11.2 (Ref. 3). On the basis of comparative deletion mapping data from rare patients with atypical deletions and a clinically similar phenotype, the TDR has been subdivided into five critical non-overlapping intervals (interval 1–5) in which at least 40 genes have been located4 (Fig. 1). The absence of a genotype–phenotype correlation in these cases and the lack of mutation in several TDR genes from patients with no
detectable 22q11.2 deletion5–11, mean that it has not been possible to identify single genes that cause the disorder. To understand the molecular basis of this disease, researchers focused their attention on animal models by creating mice with hemizygous deletions in regions of chromosome 16 that correspond to the human DGS locus12,13. In 1999, Lindsay et al.14, created the first animal model of DGS using Cre-loxP chromosome engineering in mice. They deleted a 1.2-Mb fragment (named Df1) spanning a segment of the murine chromosome 16B that is syntenic to human 22q11.2 and that contains at least 20 orthologs of genes commonly deleted in DGS/VCFS patients (Fig. 1). About 10% of mice heterozygous for this chromosomal deletion (Df1/+) showed neonatal mortality; the rest were viable and fertile. Depending on the age of examination, 18–26% of Df1/+ mice showed defective development of the
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