Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome
The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.
Vyšlo v časopise:
Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome. PLoS Genet 8(8): e32767. doi:10.1371/journal.pgen.1002861
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002861
Souhrn
The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.
Zdroje
1. EngertF, WilsonS (2011) Zebrafish neurobiology: From development to circuit function and behaviour. Dev Neurobiol
2. HutsonLD, ChienCB (2002) Wiring the zebrafish: axon guidance and synaptogenesis. Curr Opin Neurobiol 12: 87–92.
3. KrensSF, HeisenbergCP (2011) Cell sorting in development. Curr Top Dev Biol 95: 189–213.
4. LangdonYG, MullinsMC (2011) Maternal and zygotic control of zebrafish dorsoventral axial patterning. Annu Rev Genet 45: 357–377.
5. LawsonND, WolfeSA (2011) Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish. Dev Cell 21: 48–64.
6. LohrH, HammerschmidtM (2011) Zebrafish in endocrine systems: recent advances and implications for human disease. Annu Rev Physiol 73: 183–211.
7. NevinLM, RoblesE, BaierH, ScottEK (2010) Focusing on optic tectum circuitry through the lens of genetics. BMC Biol 8: 126.
8. ShepardJL, AmatrudaJF, SternHM, SubramanianA, FinkelsteinD, et al. (2005) A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc Natl Acad Sci U S A 102: 13194–13199.
9. PhillipsJB, Blanco-SanchezB, LentzJJ, TallafussA, KhanobdeeK, et al. (2011) Harmonin (Ush1c) is required in zebrafish Muller glial cells for photoreceptor synaptic development and function. Dis Model Mech 4: 786–800.
10. JurynecMJ, XiaR, MackrillJJ, GuntherD, CrawfordT, et al. (2008) Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle. Proc Natl Acad Sci U S A 105: 12485–12490.
11. Rodriguez-MariA, WilsonC, TitusTA, CanestroC, BreMillerRA, et al. (2011) Roles of brca2 (fancd1) in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish. PLoS Genet 7: e1001357 doi:10.1371/journal.pgen.1001357..
12. HuangP, XiaoA, ZhouM, ZhuZ, LinS, et al. (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29: 699–700.
13. NaseviciusA, EkkerSC (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26: 216–220.
14. DraperBW, MorcosPA, KimmelCB (2001) Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30: 154–156.
15. EisenJS, SmithJC (2008) Controlling morpholino experiments: don't stop making antisense. Development 135: 1735–1743.
16. WienholdsE, van EedenF, KostersM, MuddeJ, PlasterkRH, et al. (2003) Efficient target-selected mutagenesis in zebrafish. Genome Res 13: 2700–2707.
17. FritzA, RozowskiM, WalkerC, WesterfieldM (1996) Identification of selected gamma-ray induced deficiencies in zebrafish using multiplex polymerase chain reaction. Genetics 144: 1735–1745.
18. CarrollD (2011) Genome engineering with zinc-finger nucleases. Genetics 188: 773–782.
19. BakerM (2011) Gene-editing nucleases. Nat Methods 9: 23–26.
20. UrnovFD, RebarEJ, HolmesMC, ZhangHS, GregoryPD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11: 636–646.
21. DoyonY, McCammonJM, MillerJC, FarajiF, NgoC, et al. (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26: 702–708.
22. FoleyJE, MaederML, PearlbergJ, JoungJK, PetersonRT, et al. (2009) Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat Protoc 4: 1855–1867.
23. MengX, NoyesMB, ZhuLJ, LawsonND, WolfeSA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26: 695–701.
24. BochJ, ScholzeH, SchornackS, LandgrafA, HahnS, et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326: 1509–1512.
25. BogdanoveAJ, VoytasDF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333: 1843–1846.
26. MoscouMJ, BogdanoveAJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326: 1501.
27. MillerJC, TanS, QiaoG, BarlowKA, WangJ, et al. (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29: 143–148.
28. DengD, YanC, PanX, MahfouzM, WangJ, et al. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335: 720–723.
29. MakAN, BradleyP, CernadasRA, BogdanoveAJ, StoddardBL (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335: 716–719.
30. CermakT, DoyleEL, ChristianM, WangL, ZhangY, et al. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39: e82.
31. ChristianM, CermakT, DoyleEL, SchmidtC, ZhangF, et al. (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186: 757–761.
32. SanderJD, CadeL, KhayterC, ReyonD, PetersonRT, et al. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29: 697–698.
33. LamasonRL, MohideenMA, MestJR, WongAC, NortonHL, et al. (2005) SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310: 1782–1786.
34. StreisingerG, WalkerC, DowerN, KnauberD, SingerF (1981) Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291: 293–296.
35. StreisingerG, CoaleF, TaggartC, WalkerC, GrunwaldDJ (1989) Clonal origins of cells in the pigmented retina of the zebrafish eye. Dev Biol 131: 60–69.
36. MooreJL, RushLM, BrenemanC, MohideenMA, ChengKC (2006) Zebrafish genomic instability mutants and cancer susceptibility. Genetics 174: 585–600.
37. ParantJM, GeorgeSA, PryorR, WittwerCT, YostHJ (2009) A rapid and efficient method of genotyping zebrafish mutants. Dev Dyn 238: 3168–3174.
38. KimmelCB, BallardWW, KimmelSR, UllmannB, SchillingTF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310.
39. Westerfield M (2000) The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio). 4th ed. Eugene: Univ. of Oregon Press.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2012 Číslo 8
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