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Front Biol    2010, Vol. 5 Issue (3) : 238-245    https://doi.org/10.1007/s11515-010-0051-4
REVIEW
Advances in Drosophila gene targeting and related techniques
Zhongsheng YU1,2, Renjie JIAO1()
1. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2. Graduate School of Chinese Academy of Sciences, Beijing 100080, China
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Abstract

Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes. In Drosophila, compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest. However, applications of gene targeting have been limited because the targeting efficiency varies with different genes, and the time and labor of targeting procedure are intensive. Nevertheless, improvement of gene targeting and development of its variant technologies have received much attention of scientists. Here we review recent progress that has been made in expanding the applications of gene targeting, which include the ФC31 integration system and zinc-finger nucleases induced gene targeting, and new strategies that generate more efficient and reliable gene targeting.
Keywords gene targeting      ends-in      ends-out      ФC31 integration system      zinc-finger nucleases (ZFNs)      homologous recombination (HR)      Drosophila melanogaster     
Corresponding Author(s): JIAO Renjie,Email:rjiao@sun5.ibp.ac.cn   
Issue Date: 01 June 2010
 Cite this article:   
Zhongsheng YU,Renjie JIAO. Advances in Drosophila gene targeting and related techniques[J]. Front Biol, 2010, 5(3): 238-245.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0051-4
https://academic.hep.com.cn/fib/EN/Y2010/V5/I3/238
Fig.1  Ends-in and ends-out gene targeting in . A: Ends-in scheme. The relevant features of donor construct flanked by the elements (black boxes): FLP recombinase target (FRT) sites (green arrows), () selective marker (red boxes), I-SceI recognition sites, and I-CreI recognition sites are marked or indicated, and the asterisk represents the introduced mutation. The donor construct that contains the mutated genomic fragment is integrated into the germline by P-element-mediated transformation. A linearized targeting DNA fragment is generated by FLP and I-SceI. Recombination with the endogenous target sequence generates a tandem duplication of the targeted region. The duplication is reduced to a single copy of mutant gene after a double-stranded break (DSB) induced by I-CreI and repaired by homologous recombination, selected by the loss of . B: Ends-out scheme. The donor construct flanked by the elements, contains two stretches of homology arm (5￠ arm and 3￠ arm) interrupted by a marker that has two loxPs on both sides (brown box). After element mediated transgenesis, linearized targeting DNA is generated by FLP and I-SceI. Correct targeting events are marked by which can be removed by Cre recombinase with only a loxP site left at the target gene locus.
Fig.1  Ends-in and ends-out gene targeting in . A: Ends-in scheme. The relevant features of donor construct flanked by the elements (black boxes): FLP recombinase target (FRT) sites (green arrows), () selective marker (red boxes), I-SceI recognition sites, and I-CreI recognition sites are marked or indicated, and the asterisk represents the introduced mutation. The donor construct that contains the mutated genomic fragment is integrated into the germline by P-element-mediated transformation. A linearized targeting DNA fragment is generated by FLP and I-SceI. Recombination with the endogenous target sequence generates a tandem duplication of the targeted region. The duplication is reduced to a single copy of mutant gene after a double-stranded break (DSB) induced by I-CreI and repaired by homologous recombination, selected by the loss of . B: Ends-out scheme. The donor construct flanked by the elements, contains two stretches of homology arm (5￠ arm and 3￠ arm) interrupted by a marker that has two loxPs on both sides (brown box). After element mediated transgenesis, linearized targeting DNA is generated by FLP and I-SceI. Correct targeting events are marked by which can be removed by Cre recombinase with only a loxP site left at the target gene locus.
Fig.2  Gene targeting schemes in combination with FC31-mediated integration. A: SIRT (site-specific integrase mediated repeated targeting) strategy. The donor construct contains some of the elements that are also present in the classic gene targeting donor construct (Fig. 1). The first step of SIRT is the ends-in targeting followed by reduction to eventually place attP (blue box) in a proper site. In the second step, the integrated donor carries FRTs and P-element, as well as the desired mutation(s) (black asterisk) and attB sites (blue box). Recombination between attP and attB creates attR and attL (blue-lined empty boxes) irreversibly and a duplication of the target gene. I-CreI induced recombination that occurs at the targeting region leads to loss of the gene, and remaining of the modified target gene and attR. B: “Genomic engineering” strategy. The first step of “genomic engineering” is the ends-out targeting which replaces the target gene with a transgene that is loxP-flanked, marked and juxtaposed by an attP site. The marker is removed by Cre recombinase, leaving only the attP and the loxP at the deletion locus. In the second step, the donor construct that contains the attB site and the modified target gene and a modified target gene is integrated into the deletion locus through FC31-mediated integration. Then the extra vector sequences will be removed by Cre recombinase to generate a final engineered target gene flanked by attR and loxP sites.
Fig.2  Gene targeting schemes in combination with FC31-mediated integration. A: SIRT (site-specific integrase mediated repeated targeting) strategy. The donor construct contains some of the elements that are also present in the classic gene targeting donor construct (Fig. 1). The first step of SIRT is the ends-in targeting followed by reduction to eventually place attP (blue box) in a proper site. In the second step, the integrated donor carries FRTs and P-element, as well as the desired mutation(s) (black asterisk) and attB sites (blue box). Recombination between attP and attB creates attR and attL (blue-lined empty boxes) irreversibly and a duplication of the target gene. I-CreI induced recombination that occurs at the targeting region leads to loss of the gene, and remaining of the modified target gene and attR. B: “Genomic engineering” strategy. The first step of “genomic engineering” is the ends-out targeting which replaces the target gene with a transgene that is loxP-flanked, marked and juxtaposed by an attP site. The marker is removed by Cre recombinase, leaving only the attP and the loxP at the deletion locus. In the second step, the donor construct that contains the attB site and the modified target gene and a modified target gene is integrated into the deletion locus through FC31-mediated integration. Then the extra vector sequences will be removed by Cre recombinase to generate a final engineered target gene flanked by attR and loxP sites.
Fig.3  zinc-finger nucleases (ZFNs) induced gene targeting in . A: Diagram of the zinc-finger nucleases and its binding to the target sites. Each nuclease is composed of three zinc fingers (black balls) linked to the DNA-cleavage domain of FokI. Each finger contacts three consecutive 5￠-GNN-3￠ triplets. When both sets of fingers are bound, the cleavage domain will dimerize to form an active nuclease and cleave the spacer DNA (red bars) between the target sites. B: ZFNs induced gene targeting. The target gene is on chromosome 2, and the same chromosome carries ZFN transgenes: ZFN-A and ZFN-B (black boxes). FLP and I-SceI transgenes (black boxes) are on chromosome 3, and modified target gene is on the other chromosome 3. Upon heat shock, the ZFNs make a double-stranded break (DSB) in the target gene. Expression of FLP and I-SceI will result in a linear modified target DNA fragment. The break in the target gene will be restored to either wild type (homologous recombination (HR) with endogenous template) or result in a mutant target gene (non-homologous end joining (NHEJ) or HR with the linearized donor).
Fig.3  zinc-finger nucleases (ZFNs) induced gene targeting in . A: Diagram of the zinc-finger nucleases and its binding to the target sites. Each nuclease is composed of three zinc fingers (black balls) linked to the DNA-cleavage domain of FokI. Each finger contacts three consecutive 5￠-GNN-3￠ triplets. When both sets of fingers are bound, the cleavage domain will dimerize to form an active nuclease and cleave the spacer DNA (red bars) between the target sites. B: ZFNs induced gene targeting. The target gene is on chromosome 2, and the same chromosome carries ZFN transgenes: ZFN-A and ZFN-B (black boxes). FLP and I-SceI transgenes (black boxes) are on chromosome 3, and modified target gene is on the other chromosome 3. Upon heat shock, the ZFNs make a double-stranded break (DSB) in the target gene. Expression of FLP and I-SceI will result in a linear modified target DNA fragment. The break in the target gene will be restored to either wild type (homologous recombination (HR) with endogenous template) or result in a mutant target gene (non-homologous end joining (NHEJ) or HR with the linearized donor).
1 Adams M D, Celniker S E, Holt R A, Evans C A, Gocayne J D, Amanatides P G, Scherer S E, Li P W, Hoskins R A, Galle R F, George R A, Lewis S E, Richards S, Ashburner M, Henderson S N, Sutton G G, Wortman J R, Yandell M D, Zhang Q, Chen L X, Brandon R C, Rogers Y H, Blazej R G, Champe M, Pfeiffer B D, Wan K H, Doyle C, Baxter E G, Helt G, Nelson C R, Gabor G L, Abril J F, Agbayani A, An H J, Andrews-Pfannkoch C, Baldwin D, Ballew R M, Basu A, Baxendale J, Bayraktaroglu L, Beasley E M, Beeson K Y, Benos P V, Berman B P, Bhandari D, Bolshakov S, Borkova D, Botchan M R, Bouck J, Brokstein P, Brottier P, Burtis K C, Busam D A, Butler H, Cadieu E, Center A, Chandra I, Cherry J M, Cawley S, Dahlke C, Davenport L B, Davies P, de Pablos B, Delcher A, Deng Z, Mays A D, Dew I, Dietz S M, Dodson K, Doup L E, Downes M, Dugan-Rocha S, Dunkov B C, Dunn P, Durbin K J, Evangelista C C, Ferraz C, Ferriera S, Fleischmann W, Fosler C, Gabrielian A E, Garg N S, Gelbart W M, Glasser K, Glodek A, Gong F, Gorrell J H, Gu Z, Guan P, Harris M, Harris N L, Harvey D, Heiman T J, Hernandez J R, Houck J, Hostin D, Houston K A, Howland T J, Wei M H, Ibegwam C, Jalali M, Kalush F, Karpen G H, Ke Z, Kennison J A, Ketchum K A, Kimmel B E, Kodira C D, Kraft C, Kravitz S, Kulp D, Lai Z, Lasko P, Lei Y, Levitsky A A, Li J, Li Z, Liang Y, Lin X, Liu X, Mattei B, McIntosh T C, McLeod M P, McPherson D, Merkulov G, Milshina N V, Mobarry C, Morris J, Moshrefi A, Mount S M, Moy M, Murphy B, Murphy L, Muzny D M, Nelson D L, Nelson D R, Nelson K A, Nixon K, Nusskern D R, Pacleb J M, Palazzolo M, Pittman G S, Pan S, Pollard J, Puri V, Reese M G, Reinert K, Remington K, Saunders R D, Scheeler F, Shen H, Shue B C, Sidén-Kiamos I, Simpson M, Skupski M P, Smith T, Spier E, Spradling A C, Stapleton M, Strong R, Sun E, Svirskas R, Tector C, Turner R, Venter E, Wang A H, Wang X, Wang Z Y, Wassarman D A, Weinstock G M, Weissenbach J, Williams S M, Woodage T, Worley K C, Wu D, Yang S, Yao Q A, Ye J, Yeh R F, Zaveri J S, Zhan M, Zhang G, Zhao Q, Zheng L, Zheng X H, Zhong F N, Zhong W, Zhou X, Zhu S, Zhu X, Smith H O, Gibbs R A, Myers E W, Rubin G M, Venter J C (2000). The genome sequence of Drosophila melanogaster. Science , 287(5461): 2185-2195
doi: 10.1126/science.287.5461.2185
2 Belteki G, Gertsenstein M, Ow D W, Nagy A (2003). Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase. Nat Biotechnol , 21(3): 321-324
doi: 10.1038/nbt787
3 Beumer K, Bhattacharyya G, Bibikova M, Trautman J K, Carroll D (2006). Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics , 172(4): 2391-2403
doi: 10.1534/genetics.105.052829
4 Bibikova M, Beumer K, Trautman J K, Carroll D (2003). Enhancing gene targeting with designed zinc finger nucleases. Science , 300(5620): 764
doi: 10.1126/science.1079512
5 Bibikova M, Golic M, Golic K G, Carroll D (2002). Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics , 161(3): 1169-1175
6 Bischof J, Maeda R K, Hediger M, Karch F, Basler K (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A , 104(9): 3312-3317
doi: 10.1073/pnas.0611511104
7 Brand A H, Perrimon N (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development , 118(2): 401-415
8 Capecchi M R (1989). Altering the genome by homologous recombination. Science , 244(4910): 1288-1292
doi: 10.1126/science.2660260
9 Gao G, McMahon C, Chen J, Rong Y S (2008). A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. Proc Natl Acad Sci U S A , 105(37): 13999-14004
doi: 10.1073/pnas.0805843105
10 Golic K G, Golic M M (1996). Engineering the Drosophila genome: chromosome rearrangements by design. Genetics , 144(4): 1693-1711
11 Gong W J, Golic K G (2003). Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad Sci U S A , 100(5): 2556-2561
doi: 10.1073/pnas.0535280100
12 Greenberg A J, Moran J R, Coyne J A, Wu C I (2003). Ecological adaptation during incipient speciation revealed by precise gene replacement. Science , 302(5651): 1754-1757
doi: 10.1126/science.1090432
13 Greenspan R J. Fly pushing: the theory and practice of Drosophila genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2004
14 Grether M E, Abrams J M, Agapite J, White K, Steller H (1995). The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Genes Dev , 9(14): 1694-1708
doi: 10.1101/gad.9.14.1694
15 Groth A C, Fish M, Nusse R, Calos M P (2004). Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics , 166(4): 1775-1782
doi: 10.1534/genetics.166.4.1775
16 Hanson K D, Sedivy J M (1995). Analysis of biological selections for high-efficiency gene targeting. Mol Cell Biol , 15(1): 45-51
17 Huang J, Zhou W, Dong W, Watson A M, Hong Y (2009). From the Cover: Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering. Proc Natl Acad Sci U S A , 106(20): 8284-8289
doi: 10.1073/pnas.0900641106
18 Huang J, Zhou W, Watson A M, Jan Y N, Hong Y (2008). Efficient ends-out gene targeting in Drosophila. Genetics , 180(1): 703-707
doi: 10.1534/genetics.108.090563
19 Lankenau S, Barnickel T, Marhold J, Lyko F, Mechler B M, Lankenau D H (2003). Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products. Genetics , 163(2): 611-623
20 Liu Q, Xia Z, Zhong X, Case C C (2002). Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J Biol Chem , 277(6): 3850-3856
doi: 10.1074/jbc.M110669200
21 McCreath K J, Howcroft J, Campbell K H, Colman A, Schnieke A E, Kind A J (2000). Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature , 405(6790): 1066-1069
doi: 10.1038/35016604
22 O’Keefe L V, Smibert P, Colella A, Chataway T K, Saint R, Richards R I (2007). Know thy fly. Trends Genet , 23(5): 238-242
doi: 10.1016/j.tig.2007.03.007
23 Radford S J, Goley E, Baxter K, McMahan S, Sekelsky J (2005). Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers. Genetics , 170(4): 1737-1745
doi: 10.1534/genetics.104.036178
24 Rong Y S, Golic K G (2000). Gene targeting by homologous recombination in Drosophila. Science , 288(5473): 2013-2018
doi: 10.1126/science.288.5473.2013
25 Rong Y S, Golic K G (2001). A targeted gene knockout in Drosophila. Genetics , 157(3): 1307-1312
26 Rong Y S, Titen S W, Xie H B, Golic M M, Bastiani M, Bandyopadhyay P, Olivera B M, Brodsky M, Rubin G M, Golic K G (2002). Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev , 16(12): 1568-1581
doi: 10.1101/gad.986602
27 Rubin G M, Spradling A C (1982). Genetic transformation of Drosophila with transposable element vectors. Science , 218(4570): 348-353
doi: 10.1126/science.6289436
28 Schaefer D G, Zr?d J P (1997). Efficient gene targeting in the moss Physcomitrella patens. Plant J , 11(6): 1195-1206
doi: 10.1046/j.1365-313X.1997.11061195.x
29 Segal D J (2002). The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment. Methods , 26(1): 76-83
doi: 10.1016/S1046-2023(02)00009-9
30 Thibault S T, Singer M A, Miyazaki W Y, Milash B, Dompe N A, Singh C M, Buchholz R, Demsky M, Fawcett R, Francis-Lang H L, Ryner L, Cheung L M, Chong A, Erickson C, Fisher W W, Greer K, Hartouni S R, Howie E, Jakkula L, Joo D, Killpack K, Laufer A, Mazzotta J, Smith R D, Stevens L M, Stuber C, Tan L R, Ventura R, Woo A, Zakrajsek I, Zhao L, Chen F, Swimmer C, Kopczynski C, Duyk G, Winberg M L, Margolis J (2004). A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet , 36(3): 283-287
doi: 10.1038/ng1314
31 Thorpe H M, Smith M C (1998). In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A , 95(10): 5505-5510
doi: 10.1073/pnas.95.10.5505
32 Thyagarajan B, Olivares E C, Hollis R P, Ginsburg D S, Calos M P (2001). Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol , 21(12): 3926-3934
doi: 10.1128/MCB.21.12.3926-3934.2001
33 Venken K J, He Y, Hoskins R A, Bellen H J (2006). P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science , 314(5806): 1747-1751
doi: 10.1126/science.1134426
34 White K, Tahaoglu E, Steller H (1996). Cell killing by the Drosophila gene reaper. Science , 271(5250): 805-807
doi: 10.1126/science.271.5250.805
35 Xu T, Rubin G M (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development , 117(4): 1223-1237
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