title: Stable gene silencing in zebrafish with spatiotemporally targetable RNA interference.
abstract: The ability to regulate gene activity in a spatiotemporally controllable manner is vital for biological discovery that will impact disease diagnosis and treatment. While conditional gene silencing is possible in other genetic model organisms, this technology is largely unavailable in zebrafish, an important vertebrate model organism for functional gene discovery. Here, using short hairpin RNAs (shRNAs) designed in the microRNA-30 backbone, which have been shown to mimic natural microRNA primary transcripts and be more effective than simple shRNAs, we report stable RNA interference-mediated gene silencing in zebrafish employing the yeast Gal4-UAS system. Using this approach, we reveal at single-cell resolution the role of atypical protein kinase Cλ (aPKCλ) in regulating neural progenitor/stem cell division. We also show effective silencing of the one-eyed-pinhead and no-tail/brachyury genes. Furthermore, we demonstrate stable integration and germ-line transmission of the UAS-miR-shRNAs for aPKCλ, the expressivity of which is controllable by the strength and expression of Gal4. This technology shall significantly advance the utility of zebrafish for understanding fundamental vertebrate biology and for the identification and evaluation of important therapeutic targets.
authors: Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences, University of California, San Francisco, CA 94143-2811, USA.
authors: Zhiqiang
authors: Z
authors: Dong
authors: Jisong
authors: J
authors: Peng
authors: Su
authors: S
authors: Guo
mesh_terms: D051696:Activating Transcription Factors / Q000378:metabolism; D000818:Animals; D002455:Cell Division; D004742:Enhancer Elements, Genetic; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D000098908:Brachyury Protein; D000099293:Protein Kinase C-lambda
chemical_list: D051696:Activating Transcription Factors; D005326:Fetal Proteins; D018398:Homeodomain Proteins; D007527:Isoenzymes; D035683:MicroRNAs; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000098908:Brachyury Protein; D000099293:Protein Kinase C-lambda; C110484:tdgf1 protein, zebrafish
references: Nat Genet. 2005 Nov;37(11):1281-8
references: 16200065
references: Biochem Biophys Res Commun. 2010 Jan 15;391(3):1363-8
references: 20026059
references: Cell Death Differ. 2007 Dec;14(12):2013-20
references: 18007672
references: Mol Cell Biol. 1992 Nov;12(11):5238-48
references: 1357546
references: FEBS J. 2008 May;275(9):2177-84
references: 18384379
references: Cell. 2012 Apr 27;149(3):515-24
references: 22541426
references: RNA Biol. 2005 Jul-Sep;2(3):101-5
references: 17114924
references: Nat Genet. 2005 Nov;37(11):1289-95
references: 16200064
references: Development. 1994 Apr;120(4):1009-15
references: 7600949
references: Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19933-8
references: 21041642
references: Nat Genet. 2000 Oct;26(2):216-20
references: 11017081
references: Nat Biotechnol. 2008 Jun;26(6):695-701
references: 18500337
references: Dev Biol. 1999 Apr 15;208(2):473-87
references: 10191060
references: Genes Dev. 2002 Apr 15;16(8):948-58
references: 11959843
references: Annu Rev Biochem. 1998;67:227-64
references: 9759489
references: Nat Biotechnol. 2011 Aug 05;29(8):699-700
references: 21822242
references: Mol Cell. 2002 Jun;9(6):1327-33
references: 12086629
references: Nature. 2000 Feb 24;403(6772):901-6
references: 10706289
references: Cell. 1995 May 19;81(4):611-20
references: 7758115
references: Dev Dyn. 2010 Oct;239(10):2695-9
references: 20737511
references: Nature. 2012 Nov 1;491(7422):114-8
references: 23000899
references: Nucleic Acids Res. 2008 Dec;36(22):7100-9
references: 18988625
references: Nat Biotechnol. 2008 Jun;26(6):702-8
references: 18500334
references: Nature. 2001 May 24;411(6836):494-8
references: 11373684
references: Cell. 1998 Jan 23;92(2):241-51
references: 9458048
references: Science. 2002 Jul 5;297(5578):99-102
references: 12098699
references: Cell. 1993 Dec 3;75(5):843-54
references: 8252621
references: PLoS One. 2009 Jul 03;4(7):e6125
references: 19582161
references: Nat Biotechnol. 2011 Jan;29(1):79-83
references: 21131983
references: Nature. 1988 Oct 20;335(6192):683-9
references: 3050531
references: Anal Chem. 2010 Mar 15;82(6):2310-6
references: 20151681
references: Neuron. 2012 Apr 12;74(1):65-78
references: 22500631
references: Zebrafish. 2012 Sep;9(3):97-107
references: 22788660
references: Cell Death Differ. 2007 Dec;14(12):1998-2012
references: 18007671
references: Nat Rev Genet. 2004 Jan;5(1):69-76
references: 14708017
references: Nature. 1998 Feb 19;391(6669):806-11
references: 9486653
references: Cell. 2011 Apr 1;145(1):145-58
references: 21458673
references: Methods Mol Biol. 2009;561:41-63
references: 19504063
references: Cell. 2009 Jan 23;136(2):215-33
references: 19167326
references: Curr Biol. 2001 Oct 2;11(19):1492-502
references: 11591316
references: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13365-70
references: 19628697
references: Nat Biotechnol. 2011 Aug 05;29(8):697-8
references: 21822241
references: Nat Rev Genet. 2011 Jul 18;12(8):575-82
references: 21765459
references: Dev Biol. 2010 Jan 15;337(2):484-95
references: 19850031