Validation of shRNA clones for gene silencing in 293FT cells [Elektronische Ressource] / vorgelegt von Wen Wang

Aus dem Orthop dischen Klinik
der Universit t Wr? zburg
Orthop?disches Zentrum f r Muskoskelettale Forsc hung
Leitung: Prof. Dr. F. Jakob

Validation of shRNA clones for gene silencing
in 293FT cells


Inaugural-Dissertation
zur Erlangung der Doktorwrde der
Medizinischen Fakultt?
der
Bayerischen Julius-Maximilians-Universit t zu Wrzburg


vorgelegt von
Wen Wang
aus Anhui/PR. China


W rzburg, November 2007

?
? ii

Referent: PD Dr. Norbert Sch tze

Korreferent: Prof. Dr. med Franz Jakob

Dekan: Prof. Dr. med Matthias Frosch

Tag der m ndlichen Pr fung: 21.01.2008





















Der Promovend ist Arzt


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Wang, Wen
Orthopedic Center for Musculoskeletal Research
Orthopedic Hospital, University of W?rzburg
K nig-Ludwig-Haus, Brettreich Str. 11
D-97074 W?rzburg, Germany
Tel.: 0049 931 8031595
Fax: 0049 931 8031599

Declaration

I declare that the submitted dissertation was completed by myself and none
other, and I have not used any sources or materials other than those enclosed.

Moreover, I declare that the dissertation has not been submitted further in this
form and has not been used for obtaining any other equivalent qualification in
any other organization. Additionally, other than this degree I have not applied or
will not attempt to apply for any other degree, title or qualification in relation to
this work.

This work was completed from December 2006 to October 2007 at the Molecular
Orthopedics of Orthopaedic department, Bayerische
Julius-Maximilians-University, W?rzburg, under the supervision of PD Dr.
Norbert Sch tze (Faculty of Medicine).

Examiner: PD Dr. Norbert Sch tze
Coexaminer: Prof. Dr. Franz Jakob

Wr? zburg ................... ........................
Wen WANG



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Table of Contents

Declaration.................................................................................................ⅲ
1. Introduction............................................................................................ 1
1.1 dsRNA, siRNA, miRNA and shRNA.................................................... 1
1.1.1 dsRNA.......................................................................................... 1
1.1.2 siRNA.............................................................................. 1
1.1.3 miRNA.......................................................................................... 2
1.1.3.1 Formation and processing of miRNA.................................... 2
1.1.3.2 Cellular functions of miRNA.................................................. 4
1.1.4 shRNA......................................................................................... 5
1.2 RNA interference................................................................................ 7
1.2.1 Mechanism of RNAi..................................................................... 8
1.2.1.1 Initiation phase: dsRNA processing into siRNAs................... 8
1.2.1.2 Execution phase: assembly of siRNA containing
Silencing complexes........................................................... 11
1.2.2 Therapeutic applications of RNAi............................................... 12
1.2.2.1 Cancer................................................................................. 12
1.2.2.2 Infectious diseases................................................. 13
1.2.2.3 Neurodegenerative disorders.............................................. 14
1.2.2.4 Drawbacks of RNAi therapeutics........................... 15
1.3 Transfection...................................................................................... 16
1.3.1 Transfection by needle injection of naked DNA......................... 17
1.3.2 Transfection by physical methods.............................................. 17
1.3.2.1 Transfection by gene gun.................................................... 18
1.3.2.2 Transfection by electroporation.............................. 18
1.3.2.3 Ultrasound-facilitated transfection....................................... 19
1.3.2.4 Hydrodynamic transfection.................................... 20
1.3.3 Transfection by chemical methods............................................. 20
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1.3.3.1 Cationic lipid-mediated transfection.................................... 20
1.3.3.2 Transfection mediated by cationic polymer......................... 23
1.3.3.3 Transfection by lipid-polymer hybrid system....................... 23
1.4 293FT cell lines................................................................................ 24
1.4.1 Origins of 293 cell lines.............................................................. 24
1.4.2 Applications of 293 cell lines......................................... 26
1.5 The aim of this study......................................................................... 27
2. Materials................................................................................................ 29
2.1 shRNA bacterial glycerol stocks....................................................... 29
2.2 Cell line............................................................................... 30
2.3 Chemicals......................................................................................... 30
2.4 Experimental Kits................................................................. 31
2.5 Reagents.......................................................................................... 31
2.6 Primers................................................................................ 34
2.7 Consumables.................................................................................... 35
2.8 Apparatus.............................................................................35
3. Methods.................................................................................................37
3.1 Culturing clonal cell lines.................................................................. 37
3.2 Purification and sequencing of plasmid DNA................................... 37
3.2.1 Mini-purification of plasmid DNA................................................ 38
3.2.1.1 Cultivate and harvest bacterial cells.................................... 38
3.2.1.2 Cell lysis................................................................. 38
3.2.1.3 Clarification of lysate............................................................39
3.2.1.4 Bind DNA................................................................ 39
3.2.1.5 Wash silica membrane........................................................ 39
3.3.1.6 Dry silica membrane............................................... 39
3.2.1.7 Elute and detect highly pure DNA....................................... 39
3.2.2 Sequencing PCR....................................................................... 39
3.2.3 Clean-up of sequencing-PCR products......................................40
3.2.4 Ethanol precipitation.................................................................. 40
3.2.5 Sequencing samples in sequencer............................... 41
3.2.6 Clarify the target genes.............................................................. 41
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3.2.7 Midi-purification of plasmid DNA............................................... 41
3.2.7.1 Cultivate and harvest bacterial cells................................... 41
3.2.7.2 Cell lysis................................................................. 42
3.2.7.3 Clarification of lysate............................................................42
3.2.7.4 Bind and wash DNA............................................... 43
3.2.7.5 Elute and precipitate DNA................................................... 43
3.3 Culture 293FT cells.......................................................................... 44
3.3.1 Thawing and culture cells.......................................................... 44
3.3.2 Subculturing cells......................................................... 45
3.3.2.1 Determining viability and cell desity.................................... 45
3.3.2.2 Subculturing cells................................................................ 45
3.4 RNAi transfection by Lipofectamine 2000........................... 45
3.5 Total RNA isolation and reverse transcriptase polymerase chain
Reaction (RT-PCR) ........................................................................ 46
3.5.1 Total RNA isolation..................................................................... 46
3.5.2 cDNA synthesis............................................................ 48
3.5.3 PCR........................................................................................... 48
3.6 Gel electr