title: A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.
abstract: Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Sonal
authors.initials: S
authors.lastname: Nagarkar-Jaiswal
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Pei-Tseng
authors.initials: PT
authors.lastname: Lee
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Campbell
authors.affiliation: Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
authors.forename: Kuchuan
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Anguiano-Zarate
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Manuel Cantu
authors.initials: MC
authors.lastname: Gutierrez
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Theodore
authors.initials: T
authors.lastname: Busby
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Lin
authors.affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
authors.forename: Yuchun
authors.initials: Y
authors.lastname: He
authors.affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Schulze
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Booth
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
authors.forename: Martha
authors.initials: M
authors.lastname: Evans-Holm
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, United States.
authors.forename: Koen J T
authors.initials: KJ
authors.lastname: Venken
authors.affiliation: Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Levis
authors.affiliation: Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States.
authors.forename: Allan C
authors.initials: AC
authors.lastname: Spradling
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Hoskins
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
authors.forename: Hugo J
authors.initials: HJ
authors.lastname: Bellen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000378:metabolism; D007858:Learning / Q000502:physiology; D018613:Microscopy, Confocal; D016254:Mutagenesis, Insertional; D034622:RNA Interference; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D051178:alpha Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: C510146:BRP protein, Drosophila; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D025521:Tumor Suppressor Proteins; D051178:alpha Catenin; C070350:dlg1 protein, Drosophila; D049452:Green Fluorescent Proteins
keywords: D. melanogaster; RMCE; bruchpilot; cell biology; deGradFP; discs large 1; dunce; iGFPi; neuroscience
references.citation: BMC Genomics. 2009;10:249
references.pmid: 19476619
references.citation: Genesis. 2002 Sep-Oct;34(1-2):1-15
references.pmid: 12324939
references.citation: Nucleic Acids Res. 2014 Jan;42(Database issue):D780-8
references.pmid: 24234449
references.citation: Nucleic Acids Res. 2008 Oct;36(18):e114
references.pmid: 18676454
references.citation: J Comp Neurol. 1998 Dec 14;402(2):197-209
references.pmid: 9845243
references.citation: PLoS Genet. 2014 Aug;10(8):e1004540
references.pmid: 25101996
references.citation: Genetics. 2007 Mar;175(3):1505-31
references.pmid: 17194782
references.citation: Nat Struct Mol Biol. 2012 Jan;19(1):117-21
references.pmid: 22157958
references.citation: J Cell Biol. 2008 Apr 7;181(1):157-70
references.pmid: 18391075
references.citation: Neuron. 1991 Mar;6(3):455-67
references.pmid: 1848082
references.citation: Cell Rep. 2012 Oct 25;2(4):1014-24
references.pmid: 23063361
references.citation: Genetics. 2013 Feb;193(2):421-30
references.pmid: 23150604
references.citation: Biol Open. 2012 May 15;1(5):488-97
references.pmid: 23213441
references.citation: Methods Cell Biol. 1994;44:635-54
references.pmid: 7707973
references.citation: Genetics. 2011 Jul;188(3):731-43
references.pmid: 21515576
references.citation: Nat Cell Biol. 2013 Mar;15(3):261-73
references.pmid: 23417122
references.citation: Genes Dev. 1994 Mar 1;8(5):598-613
references.pmid: 7523244
references.citation: Nature. 2011 Mar 24;471(7339):527-31
references.pmid: 21430782
references.citation: Dev Biol. 1988 Jun;127(2):392-407
references.pmid: 3132409
references.citation: J Neurosci. 2012 Nov 21;32(47):16586-96
references.pmid: 23175814
references.citation: J Comp Physiol A. 1985 Sep;157(2):263-77
references.pmid: 3939242
references.citation: Nat Genet. 2004 Mar;36(3):283-7
references.pmid: 14981521
references.citation: Proc Natl Acad Sci U S A. 1987 Nov;84(21):7433-7
references.pmid: 3118368
references.citation: Genetics. 2013 Feb;193(2):411-9
references.pmid: 23150601
references.citation: Dev Biol. 1972 Jan;27(1):71-83
references.pmid: 4621757
references.citation: Cell. 1991 Aug 9;66(3):451-64
references.pmid: 1651169
references.citation: Neuron. 2006 Mar 16;49(6):833-44
references.pmid: 16543132
references.citation: Oncogene. 1999 Jan 7;18(1):219-32
references.pmid: 9926937
references.citation: Cell Rep. 2012 Oct 25;2(4):1002-13
references.pmid: 23063363
references.citation: Chromosome Res. 2010 Apr;18(3):307-24
references.pmid: 20213139
references.citation: Methods Mol Biol. 2012;859:203-28
references.pmid: 22367874
references.citation: J Cell Biol. 1996 Sep;134(6):1469-82
references.pmid: 8830775
references.citation: Cell Rep. 2012 Oct 25;2(4):991-1001
references.pmid: 23063364
references.citation: Learn Mem. 1998 May-Jun;5(1-2):102-14
references.pmid: 10454375
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):15948-53
references.pmid: 21896744
references.citation: G3 (Bethesda). 2014 Dec;4(12):2381-7
references.pmid: 25298537
references.citation: Science. 2006 May 19;312(5776):1051-4
references.pmid: 16614170
references.citation: Cell. 2013 Jul 3;154(1):228-39
references.pmid: 23827685
references.citation: Nat Methods. 2009 Jun;6(6):435-7
references.pmid: 19465918
references.citation: Genetics. 2007 Mar;175(3):1089-104
references.pmid: 17179094
references.citation: Nucleic Acids Res. 2015 Apr 30;43(8):e56
references.pmid: 25712101
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15050-5
references.pmid: 11742088
references.citation: J Cell Biol. 1981 Jul;90(1):101-7
references.pmid: 6265472
references.citation: Nat Methods. 2011 Sep;8(9):737-43
references.pmid: 21985007
references.citation: J Neurosci. 2008 Jan 2;28(1):304-14
references.pmid: 18171947
references.citation: J Cell Sci. 2012 Jan 1;125(Pt 1):233-45
references.pmid: 22266901
references.citation: Development. 2005 Jun;132(12):2743-58
references.pmid: 15901661
references.citation: Neuron. 2014 Jan 22;81(2):280-93
references.pmid: 24462095
references.citation: Neuron. 2007 Jan 18;53(2):201-15
references.pmid: 17224403
references.citation: Dev Biol. 1994 Oct;165(2):716-26
references.pmid: 7958432
references.citation: Development. 2013 Jun;140(11):2434-42
references.pmid: 23637332
references.citation: Science. 2000 Mar 24;287(5461):2185-95
references.pmid: 10731132
references.citation: Neuron. 2001 Nov 8;32(3):415-24
references.pmid: 11709153
references.citation: Cell Rep. 2015 Mar 3;10(8):1410-21
references.pmid: 25732830
references.citation: J Mol Biol. 1991 Dec 5;222(3):553-65
references.pmid: 1660926
references.citation: Fly (Austin). 2010 Jan-Mar;4(1):53-9
references.pmid: 20139712
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13794-9
references.pmid: 21808003
references.citation: Genes Dev. 1997 May 1;11(9):1122-31
references.pmid: 9159393
references.citation: Dev Biol. 2008 Jun 1;318(1):1-16
references.pmid: 18423436
references.citation: Cell. 2009 Sep 4;138(5):947-60
references.pmid: 19737521
references.citation: Int Rev Neurobiol. 2006;75:323-65
references.pmid: 17137935
references.citation: Genetics. 2005 Oct;171(2):571-81
references.pmid: 15972463
references.citation: Neuron. 2011 Oct 20;72(2):202-30
references.pmid: 22017985
references.citation: Genetics. 2012 Mar;190(3):931-40
references.pmid: 22174071
references.citation: Science. 2003 Dec 5;302(5651):1765-8
references.pmid: 14657498