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本帖最后由 qianqianlaile 于 2011-4-18 20:59 编辑
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3 c8 ]$ }; K' A: p' M. XA Rapid and Scalable System for Studying Gene Function in Mice Using Conditional RNA Interference
& k1 C+ l, e% F G) Z, v) R9 G6 NPrem 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' s: k6 R+ d# Q" t: v. y
( U. @$ @" q% \ T( J+ P2 g2 The Watson School of Biological Sciences, Cold Spring Harbor, NY 11724, USA# x# ]7 c3 _1 |9 E; P0 O
+ q/ O$ j; t$ [$ k1 c3 Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA! \ r$ Q- a6 r
/ x7 ?, c5 }/ F6 }2 q [, f4 Medical Scientist Training Program, Stony Brook University Medical Center, Stony Brook, New York 11794, USA1 f5 N; V5 y! t8 Y9 N( B @- }
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5 Department of Pathology, Stony Brook University Medical Center, Stony Brook, New York 11794, USA
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4 |- G2 y" ]- j6 Molecular Medicine Division, Walter & Eliza Hall Institute of Medical Research, Parkville 3052, Australia
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7 Helen Diller Family Comprehensive Cancer Center and Department of Laboratory Medicine, University of California, San Francisco, CA, USA1 F! }: B" [$ u
& q1 p3 N( s0 J5 C# UReceived 10 June 2010; revised 17 December 2010; accepted 5 March 2011. Published: March 31, 2011. Available online 31 March 2011. 9 q" }: ]# O, h# b
& ]( R- o6 {- Z xSummary4 p$ V3 U. N2 H& j
RNA 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.
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PaperClip
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2 V0 i+ B- V6 }2 V7 {Graphical Abstract$ J: s _2 {( m" g* j& [& @/ `3 b3 @
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Highlights3 R. _$ ?, c5 I! X
► 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( j: S: H; K) W# r3 N, D* y
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Article Outline
& s+ |* c8 v. o. V6 l1 e; _7 iIntroduction) T3 @2 I: n- C3 i# S0 F9 y5 P2 h7 c
Results+ @( J3 w" A9 i8 g, ]3 H
Generation of a Targeting Construct
3 m2 h" i9 t( Q [, [ColA1-Targeted shRNAs Enable Potent and Reversible Knockdown in ES Cells
b8 H8 F/ |$ U7 Q; I m6 iColA1-Targeted shRNAs Enable Potent and Reversible Gene Suppression In Vitro
+ E$ q: b! M* e' e: dTransgenic shRNAs Do Not Impair Normal MicroRNA Processing+ \9 W! U5 W$ N1 v" W! p
ColA1-Targeted shRNAs Enable Potent and Reversible Gene Suppression In Vivo
. V# q N7 e1 fA Tet-Transactivator Line that Enables Efficient Knockdown in a Broad Range of Adult Tissues5 a/ p" r, d- ~: Q$ Z$ V
Studying Gene Function during Embryonic and Postnatal Development: Reversible and Irreversible Consequences of Hyperactive Wnt Signaling: d: Z1 t8 R( ]9 X5 L
Studying Gene Function in Disease Initiation and Maintenance: APC Suppression Is Required for the Initiation and Maintenance of T-ALL
5 r" x c- T4 O s7 C# e( aStudying Genetic Modifiers of Disease: p19ARF Suppression Promotes the Initiation and Maintenance of KrasG12D-Induced Lung Cancer0 B/ b j( a& I. H& x3 T8 ]" C/ M
Accelerating the Analysis of Complex Models of Disease Using Conditional RNAi
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Experimental Procedures
: a* U: G0 q$ V3 L1 C6 N5 k7 zTargeting Constructs and ES Cell Targeting
+ e0 w* J) b i: @- m7 U5 F: lTransgenic and Speedy Mice
' F% W* _, D. G( r! b* @% H; ]Alcian Blue and Alizarin Red Skeletal Staining
$ R P, _4 q* E! N1 I k% o+ f( ~: |Lymphoma Transplants and Monitoring
$ E7 w- I3 I7 {+ s7 }2 \& A! vCell Culture and Expression Analysis; Y8 i9 X' ~( K1 J* C8 ~1 n- k
RNA Extraction and Quantitative Real-Time PCR
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( o w6 U+ ~+ a q# }这篇文章是要付费的，所以摘要之外的部分只有提纲 |
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发表于 2011-4-18 20:59
发表于 2011-4-19 09:18
