Versatile extensions of the Flp-recombinase-mediated cassette exchange technology [Elektronische Ressource] : RMCE multiplexing approaches meet the needs for predictable genome engineering / Sören Turan. Institut Experimentelle Hämatologie der Medizinische Hochschule Hannover. Betreuer: Jürgen Bode ; Beate Sodeik

Medizinische Hochschule Hannover
Experimentelle Hämatologie


Versatile Extensions of the Flp-Recombinase-Mediated

Cassette Exchange Technology: RMCE multiplexing
approaches meet the needs for predictable genome
engineering

INAUGURAL - DISSERTATION
Zur Erlangung des Grades eines Doktors der
Naturwissenschaften
-Doctor rerum naturalium-
(Dr. rer. nat.)


vorgelegt von



Sören Turan





geboren am 23.11.1981 in Braunschweig

Hannover 2010



Angenommen vom Senat der Medizinischen Hochschule Hannover am 04.03.2011
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover

Präsident: Prof. Dr. med. Dieter Bitter-Suermann
Wissenschaftliche Betreuung: Prof. Dr. rer. nat. Jürgen Bode
Wissenschaftliche Zweitbetreuung: Prof. ‘in. Dr. rer. nat. Beate Sodeik


1. Gutachter: Prof. Dr. rer. nat. Jürgen Bode
2. Gutachterin: Prof.‘in Dr. rer. nat. Beate Sodeik
3. Gutachter: Prof. Dr. rer. nat. Andreas Kispert



Tag der mündlichen Prüfung vor der Prüfungskommission: 04.03.2011

Prof. Dr. rer. nat. Achim Gossler
Prof. Dr. rer. nat. Jürgen Bode
Prof.‘in Dr. rer. nat. Beate Sodeik
Prof. Dr. rer. nat. Andreas Kispert

Publication list

Publication list
Parts of this dissertation were published (see below).
Turan S., Kuehle J., Schambach A., Baum C., Bode J., Multiplexing RMCE:
Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange
thTechnology, Journal of Molecular Biology, 402:52-69, ISSN 0022-2836, 10
September, 2010

Conference contribution

Turan S., Heinz N., Schambach A. and Bode J., Chromosome-based vectors:
thNovel routes to predictable gene expression; Poster presentation; 18 Annual
thCongress of the European Society for Gene and Cell Therapy (ESGCT), October 22
th- 25 , 2010, Milano

Turan S., Kuehle J., Schambach A. and Bode J., Novel heterospecific FRTs
Enable Gene Targeting of Several Loci by Flp-Multiplex-RMCE; Poster presentation;
th17 Annual Meeting of the German Society for Gene Therapy (DG GT e.V.), October
th th7 - 9 ,2010, Munich
Turan S., Kuehle J., Schambach A. and Bode J., RMCE Multiplexing:
simultaneous or successive targeting of independent genomic sites; Poster
presentation; Second Annual Protein & Antibody Engineering Summit (PEGS)
th thEurope, October 5 – 7 , 2010, Hannover

Turan S., Kuehle J., Schambach A. and Bode J., Multiplexing RMCE: Versatile
Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology;
Poster presentation; International Summer School ―Gene regulation, cell
th thdifferentiation and tolerance‖, September 12 - 16 , 2010; Goslar


Table of content

Table of Content
i
Zusammenfassung .................................................................................................. vi
Summary .................................................................................................................viii
1 Introduction ........ 1
1.1 Recombination ............................................................................................... 1
1.2 Homologous recombination ........................................... 1
1.3 Site specific recombination ............................................ 2
1.3.1 Serine and Tyrosine recombinases ......................................................... 5
1.3.2 Recombination system Cre/loxP ............................. 5
1.3.3 Recombination system Flp/FRT .............................. 6
1.3.3.1 Modification of Flp .......................................................................... 10
1.4 Modification of the eukaryotic genome ........................ 10
1.4.1 Gene targeting by homologous recombination...... 11
1.4.2 Generation of conditional ‗knock out‘ mice with the help of SSR .......... 12
1.4.3 FlEx is a genetic switch to monitor correct gene ablation in conditional
‗knock out‘ mice .................................................................................... 12
1.4.4 Gene Trap combined with the FlEx-ing principle ................................... 13
1.5 Recombinase-mediated cassette exchange (RMCE) .. 14
1.5.1 Cre- and ΦC31-RMCE .......................................... 16
1.6 Multiplexing-RMCE ...................................................... 17
1.7 Aims of the project ....................................................... 18
2 Results .............................................. 20
2.1 Pre-test (PCR) ............................................................. 20
2.1.1 Construction of novel mutant FRTs ....................... 21
2.1.2 Characterisation of novel mutant FRTs ................................................. 23
2.2 Transient RMCE .......................................................... 26
2.2.1 Vector architecture for Transient RMCE ............... 26
2.2.2 Transient RMCE of novel mutant FRT pairs ......................................... 28
2.3 Multiplexing-RMCE ...................................................... 33
2.3.1 Genomic RMCE with novel FRTs (one target) ...... 34
2.3.2 Construction of multiplexing-RMCE competent cell lines ...................... 36
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2.3.3 Multiplexing-RMCE with clone O5 ......................................................... 38
2.3.4 Targeted clones show double expression of fluorescent markers ......... 46
3 Discussion ........................................................................................................ 48
3.1 Construction of valid novel FRTs has to meet three criteria ........................ 48
3.1.1 48 bp FRTs rather than 34 bp minimal sites are required for RMCE .... 48
3.1.2 A functional mutant FRT is defined by three structural features as
proposed by Umlauf & Cox, (1988) ....................................................... 49
3.1.3 The ―4 bp rule‖ revealed by Schlake & Bode, (1994) is necessary for
obligate heterospecifity between FRTs ................. 49
3.2 Characterization of novel FRTs ................................................................... 50
3.2.1 The PCR test permits detection of recombinational activity among
homologous and heterospecific FRTs ................................................... 50
3.2.2 Mutants F , F and F are functional and safe ................................... 52 13 14 15
3.2.3 Transient RMCE: A practical tool for development and elucidation of
novel RMCE vectors ............................................. 52
3.2.4 Transient RMCE results are in favor of combinations F -F and F -F 13 14 14 15
.............................................................................. 54
3.3 Genomic RMCE ........................................................... 55
3.3.1 Sets F -F and F -F are both capable of genomic RMCE in a 11 12 13 14
polyclonal cell line ................................................. 55
3.4 Multiplexing-RMCE ...................................................... 57
3.4.1 Transfection and sorting steps are limiting factors for multiplexing-RMCE
.............................................................................. 57
3.4.2 Targeting both loci of O5 is feasible ...................... 58
3.4.2.1 Negative selection of targeted clone O5 with Ganciclovir is dose-
dependent............................................................................................ 59
3.4.3 PCR as well as Southern blot analyses prove double exchange in
subclones of O5 .................... 60
3.4.4 Targeting both loci of M28 is not feasible .............................................. 61
3.5 Cre and Flp in the context of (multiplexing)-RMCE ...... 61
3.5.1 Comparison of mutant recognition sites indicates enhanced promiscuity
and a lower self-recognition potential of lox sites .................................. 62
3.5.2 loxP pseudo sites in various genomes interfere with multiplexing-Cre-
RMCE ................................................................... 64
3.6 Perspectives ................................ 66
3.6.1 Enhancing efficiency of multiplexing-RMCE.......... 66
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3.6.2 Combined ―polyclonal targeting‖ and ―promoter-outside‖ strategies
simplify characterization of clones and prevent random integration of
donors ................................................................................................... 69
3.6.3 RMCE with several heterospecific sites ................ 70
3.6.4 European Conditional Mouse Mutagenesis (EUCOMM) program:
generation of targetable pseudo homozygous mice .............................. 72
3.6.5 (Multiplexing)-RMCE in ES and iPS cells .............................................. 74
4 Material & Methods .......................................................... 76
4.1 Instrumentation, Chemicals, Enzymes and Computer Programs ................ 76
4.1