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本帖最后由 qianqianlaile 于 2011-4-18 20:59 编辑
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A Rapid and Scalable System for Studying Gene Function in Mice Using Conditional RNA Interference
' _- C( \7 J* [- W( _1 v7 q0 L- lPrem K. Premsrirut1, 4, 8, Lukas E. Dow1, 8, Sang Yong Kim1, Matthew Camiolo1, 4, Colin D. Malone1, 2, Cornelius Miething1, Claudio Scuoppo1, 2, Johannes Zuber1, Ross A. Dickins1, 6, Scott C. Kogan7, Kenneth R. Shroyer5, Raffaella Sordella1, Gregory J. Hannon1, 3 and Scott W. Lowe1, 3, ,
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1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA( h. W0 A+ N |( e7 F
2 m1 R. o5 N1 V2 The Watson School of Biological Sciences, Cold Spring Harbor, NY 11724, USA6 H5 H4 _, H d8 E& w$ X2 a
6 |1 V' P! a$ Q3 Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA. I7 x% J. a7 o' D1 g- t7 y1 r( U
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4 Medical Scientist Training Program, Stony Brook University Medical Center, Stony Brook, New York 11794, USA$ a) A8 U$ J' v8 l) H
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5 Department of Pathology, Stony Brook University Medical Center, Stony Brook, New York 11794, USA/ _# G6 G- ?/ H8 q! q# }6 M
/ j, n# }5 n3 x7 u8 A& w: w9 P2 E6 Molecular Medicine Division, Walter & Eliza Hall Institute of Medical Research, Parkville 3052, Australia4 }; P. i4 G2 \% [( y, J
8 c7 {4 v' x* ^6 M7 Helen Diller Family Comprehensive Cancer Center and Department of Laboratory Medicine, University of California, San Francisco, CA, USA
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Received 10 June 2010; revised 17 December 2010; accepted 5 March 2011. Published: March 31, 2011. Available online 31 March 2011. ( b% t& f' J# {5 g5 Q3 ~' b6 j
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Summary
3 e- ?0 O& d& y, oRNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene.1 s8 v1 Q; |! f* W% y
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0 |9 ?, _7 U/ l$ t* bGraphical Abstract" ~9 J7 z9 n8 m* x
4 j1 k: I. @' l5 t/ K# A1 jHighlights" F& l; h( X. o: \# J. q
► shRNA transgenics enable potent and reversible fluorescence-marked gene silencing ► Transgenic mouse production is fast, efficient, and scalable ► “Speedy” ES cells accelerate the evaluation of gene function in mouse models ► Reversible gene suppression can pinpoint pathways for therapeutic intervention
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Article Outline
" t+ P8 \! Y7 uIntroduction
$ K4 ?9 `+ ~. b5 h. G+ D- A& EResults2 O- n& U* n. E( P3 P& o/ n
Generation of a Targeting Construct
5 h3 T( s$ g5 A lColA1-Targeted shRNAs Enable Potent and Reversible Knockdown in ES Cells
: i( ?9 [0 k3 ^( t5 K7 [- wColA1-Targeted shRNAs Enable Potent and Reversible Gene Suppression In Vitro
7 i: |% I: Q4 ~+ kTransgenic shRNAs Do Not Impair Normal MicroRNA Processing
% P$ D( ^3 k9 [* p- v3 l I5 A' r) MColA1-Targeted shRNAs Enable Potent and Reversible Gene Suppression In Vivo8 x" c, q% f* |1 v
A Tet-Transactivator Line that Enables Efficient Knockdown in a Broad Range of Adult Tissues
1 d" A6 K+ N$ o( NStudying Gene Function during Embryonic and Postnatal Development: Reversible and Irreversible Consequences of Hyperactive Wnt Signaling
8 C. y, T6 f5 j/ Z3 {, MStudying Gene Function in Disease Initiation and Maintenance: APC Suppression Is Required for the Initiation and Maintenance of T-ALL) L. {" T' V% w# G e7 f
Studying Genetic Modifiers of Disease: p19ARF Suppression Promotes the Initiation and Maintenance of KrasG12D-Induced Lung Cancer
/ p9 R; Z- f% XAccelerating the Analysis of Complex Models of Disease Using Conditional RNAi
* Q) O& G9 K9 Y& a/ C9 fDiscussion
+ V8 w8 P5 d3 n% s8 ^+ k* mExperimental Procedures: p5 K w% Y7 P6 F# [& w
Targeting Constructs and ES Cell Targeting% G+ H" k; T" R2 q+ y
Transgenic and Speedy Mice s, \. h& g. ^6 P
Alcian Blue and Alizarin Red Skeletal Staining
; T/ d0 G- H6 }Lymphoma Transplants and Monitoring
/ b% `' v- A& m6 V4 _! @; {, eCell Culture and Expression Analysis
0 ]) v) f- g2 V/ G: @3 qRNA Extraction and Quantitative Real-Time PCR' P! N+ ~4 q( ^& r/ N" x# ?' k5 r
Lung Histopathology and Immunofluorescence
7 Y/ l5 H+ p7 H5 O这篇文章是要付费的,所以摘要之外的部分只有提纲![](static/image/smiley/default/smile.gif) |
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