Cet ouvrage et des milliers d'autres font partie de la bibliothèque YouScribe
Obtenez un accès à la bibliothèque pour les lire en ligne
En savoir plus

Partagez cette publication











Establishment of in vivo bioluminescence imaging
models for tumor immunology








InauguralDissertation
Tewfik Miloud
























































DISSERTATION


Submittedtothe
FakultätfürBiowissenschaftenof
RuprechtKarlUniversität
Heidelberg

andtothe

UFRsciencesdelavieof
UniversitédeBourgogne
Dijon



Presentedby
Tewfik Miloud
Bornin:LeCreusot,France
Oralexamination:23rdofNovember2007







Establishment of in vivo bioluminescence imaging
models for tumor immunology












Supervisors:
Prof.Dr.GünterJ.Hämmerling
Prof.JohannaChluba

Reviewers:
Dr.Protzer
Dr.Aprahamian
Table of contents

List of figures: .................................................................................................... vii

List of tables: ..................................................................................................... viii

Publications: ........................................................................................................ ix

Summary .............................................................................................................. x
A. Introduction .................................................................................. 1
1. Imaging.........................................................................................................2
1.1 Tumor imaging in mouse model of cancer...................................................................2
1.1.1 MRI................................................................................................................................................2
1.1.2 CT...................................................................................................................................................3
1.1.3 US...................................................................................................................................................3
1.1.4 PET and SPECT............................................................................................................................3
1.2 Optical imaging...............................................................................................................4
1.2.1 Bioluminescence imaging..............................................................................................................5
1.2.1.1 Bioluminescence.....................................................................................................................5
1.2.1.2 Principle of Bioluminescence imaging..................................................................................5
1.2.2 Luciferases.....................................................................................................................................6
1.2.2.1 Firefly luciferase.....................................................................................................................6
1.2.2.2 Renilla luciferase....................................................................................................................7
1.2.2.3 Bacterial luciferase (lux operon)............................................................................................7
1.2.3 Light transmission through mammalian tissue..........................................................................7
1.2.4 Use of BLI in monitoring biological processes............................................................................8

2. Animal models for tumor immunology.....................................................9
2.1 Transplantable tumor models........................................................................................9
2.2 Autochthonous tumor models......................................................................................10
2.2.1 SV40-driven transgenic models.................................................................................................11
2.2.2 Inducible gene expression systems.............................................................................................11
2.2.2.1 Cre/LoxP system...................................................................................................................12
2.2.2.2 Conditional liver tumor model..............................................................................................15



i Table of contents
3. Bacteria and anticancer therapies...........................................................15
3.1 Bacteria and tumor colonization.................................................................................15
3.2 Why do bacteria colonize tumors?..............................................................................16
3.3 Bacteria as a vaccine vehicle........................................................................................17

4. Aims of the study.......................................................................................18
B. Material and methods ................................................................ 19
1. Material......................................................................................................20
1.1 Chemicals.......................................................................................................................20
1.2 Basic equipment............................................................................................................20
1.3 Kits.................................................................................................................................20
1.4 Technical devices...........................................................................................................21
1.5 Buffers and solutions....................................................................................................22
1.6 Media..............................................................................................................................25
1.6.1 Media for bacterial culture........................................................................................................25
1.6.2 Media for cell culture..................................................................................................................25
1.7 Bacterial strains............................................................................................................26
1.8 Mammalian cell lines....................................................................................................26
1.9 Mouse lines....................................................................................................................26
1.10 Antibodies......................................................................................................................27
1.11 Plasmids.........................................................................................................................27
1.12 PCR primers..................................................................................................................28

2. Methods......................................................................................................29
2.1 Molecular biology.........................................................................................................29
2.1.1 Bacterial culture..........................................................................................................................29
2.1.2 Preparation of CaCl competent (heat competent) E. coli bacteria........................................292
2.1.3 Bacteria storage...........................................................................................................................29
2.1.4 Transformation of heat (CaCl ) competent E. coli bacteria....................................................292
2.1.5 DNA minipreparation (alkaline lysis).......................................................................................30
2.1.6 DNA midipreparation (alkaline lysis).......................................................................................30
2.1.7 Restriction digestion...................................................................................................................31
2.1.8 Dephosphorylation of DNA ends (e.d. in vectors for ligation).................................................31
ii Table of contents
2.1.9 Electrophoretic separation of DNA fragments in agarose gels...............................................31
2.1.10 Isolation of DNA fragments from agarose gels with Qiaquick Gel extraction kit...............32
2.1.11 Ligation of DNA fragments and vectors..................................................................................32
2.1.12 PCR (Polymerase Chain Reaction)..........................................................................................32
2.1.12.1 Analytical PCR......................................................................................................................33
2.1.12.2 Preparative PCR...................................................................................................................33
2.1.14 Preparation of DNA microinjection........................................................................................34
2.1.15 Preparation of BAC DNA for transgenic mice production...................................................34
2.1.15.1 Preparation of electrocompetent cells and generation of recombinants.............................34
2.1.15.2 Transformation and modification of RCPI23-14C7............................................................35
2.1.15.3 Purification of BAC DNA for microinjection......................................................................36
2.2 Mouse work...................................................................................................................37
2.2.1 Production of transgenic mice....................................................................................................37
2.2.1.1 Layout of a microinjection instrument.................................................................................37
2.2.1.2 Preparation of glass capillary needles..................................................................................37
2.2.1.3 Preparation of zygotes from pregnant mice.........................................................................37
2.2.1.4 Microinjection of the transgene cassettes............................................................................38
2.2.1.5 Transfer of embryos into oviduct.........................................................................................38
2.2.2 Typing of transgenic mice..........................................................................................................38
2.2.2.1 Isolation of tail DNA.............................................................................................................38
2.2.2.2 Mouse genotyping by non-radioactive Southern blot analysis...........................................38
2.2.3 Lymphocyte preparation from blood and genotyping by FACS............................................39
2.2.4 Splenocyte preparation...............................................................................................................39
2.2.5 Tumor inoculation.......................................................................................................................40
2.2.6 Bioluminescence imaging of mice..............................................................................................40
2.2.7 Measurement of alanine aminotransferase activity in mouse plasma....................................40
2.3 Cell culture....................................................................................................................41
2.3.1 Long term storage.......................................................................................................................41
2.3.2 Cell transfection..........................................................................................................................41
2.3.3 FACS measurement....................................................................................................................42
2.3.4 Luciferase assay...........................................................................................................................42
2.3.6 Western blot................................................................................................................................43
2.3.6.1 Precipitation of proteins from bacterial supernatant..........................................................43
2.3.6.2 Cell lysate..............................................................................................................................43
2.3.6.3 SDS-Polyacrylamide (PAA) Gel Electrophoresis................................................................44
2.3.6.4 Electrotransfer of proteins from SDS-PAGE gel to membrane..........................................44
2.3.6.5 Immunoblotting....................................................................................................................45

iii Table of contents
C. Results .......................................................................................... 46
1. Selection of an optimal luciferase for BLI..............................................47
1.1 Generation of eGFP-2a-Luciferase constructs...........................................................47
1.1.1 Cloning of the 2A constructs......................................................................................................49
1.2 2A cleavage and generation of stable clones...............................................................51
1.3 Luciferase comparison in vitro.....................................................................................54
1.4 Luciferase comparison in vivo.....................................................................................55

2. BLI to follow tumor growth in mice........................................................58
2.1 Monitoring of tumorigenesis and metastasis in vivo..................................................58
2.1.1 Antigen presentation...................................................................................................................58
2.1.2 Sensitivity of detection................................................................................................................60
2.1.3 Monitoring of tumor growth by BLI.........................................................................................62
2.1.3.1 Subcutaneous tumor growth.................................................................................................62
2.1.3.2 In vivo analysis of metastasis colonization..........................................................................63
2.2 Generation of autochthonous hepatocarcinoma model for BLI...............................64
2.2.1 Generation and characterization of ASC mice.........................................................................64
2.2.2 Construction of the Stop-CBGr99-FlpAmp cassette................................................................65
2.2.3 Modification of the albumin gene containing BAC..................................................................65
2.2.4Screening of founder mice............................................................................................68
2.2.5Characterization of ASC and ASCT mice..................................................................69
2.2.5.1 Unrecombinedmice...............................................................................................................69
2.2.5.2 Recombinedmouse................................................................................................................70

3. Bioluminescence imaging of tumors in live animals with bacteria
encoding luciferase and their usage in tumor therapy..................................75
3.1 Visualization of tumors................................................................................................75
3.1.1 Subcutaneous model...................................................................................................................75
3.1.1.1 V.choleraecolonization.........................................................................................................75
3.1.1.2 L!arabinose!inducibleexpression..........................................................................................77
3.1.1.3 Top10.luxtumorcolonization................................................................................................79



iv Table of contents
3.2 Bacteria secreting interleukin......................................................................................81
3.2.1 The Hly secretion system............................................................................................................81
3.2.3 GM-CSF construct......................................................................................................................83
3.2.4 IL-2 construct..............................................................................................................................83
3.2.5 Secretion of interleukin in Top10 bacteria................................................................................84
3.2.6 Tumor studies with GM-CSF secreting E.coli..........................................................................84
3.3 Spontaneous tumor model............................................................................................86
3.3.1 Alb-Tag model.............................................................................................................................86
3.3.2 RIP-Tag-5 model.........................................................................................................................88
3.3.3 Her2-Neu model..........................................................................................................................89

4. Generation of a conditional luciferase reporter mouse.........................91
4.1 Generation and Characterization of ßactin-Fstop-eGFP-2a-CBGr99 mouse line.91
4.1.1 Construct.....................................................................................................................................91
4.1.2 Screening of founder mice..........................................................................................................92
4.1.3 Characterization of ßactin-eGFP-2A-CBGr99 mice................................................................92
4.2 Generation and characterization of CAG-CBGr99-2A-mCherry mouse line........95
4.2.1 Construct.....................................................................................................................................95
4.2.2 Screening of founder mice..........................................................................................................95
4.2.3 Characterization of CAG-CBGr99-2A-mCherry mice............................................................96
D. Discussion .................................................................................... 99
1. Selection of an optimal luciferase for BLI............................................102

2. Generation of autochthonous hepatocarcinoma model for BLI.........104

3. Bioluminescence imaging of tumors in live animals with bacteria
encoding luciferase and their usage in tumor therapy................................108

4. Generation and characterization of ß-actin-fstop-eGFP-2a-CBGr99
mouse line.........................................................................................................112

v Table of contents
E. Conclusion ................................................................................. 115
F. References .................................................................................. 117
Abbreviation list: ............................................................................................. 128

Acknowledgements .......................................................................................... 131

vi

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin