1 Introduction ................................................. 4
1.1 Mouse models for tumor immunology research .............. 4
1.1.1 Oncogene transgenic mouse models ................. 4
1.1.2 SV40 Large T antigen as transgenic oncogene in
mouse models ..................................... 5
1.1.3 Immunological characteristics of Tag ............. 5
1.1.4 Conditional mouse tumor models ................... 6
1.1.5 Controlled tumor development following Cre4oxP
mediated recombination ........................... 7
1.1.6 Leakiness of LoxP-flanked attenuators ............ 9
1.1.7 The LoxP-Tag transgenic mouse model .............. 9
1.2 Small Animal Imaging ................................... 11
1.2.1 Firefly luciferase for in vivo imaging .......... 13
1.2.2 Mouse models for bioluminescent imaging ......... 14
1.3 Adoptive T cell therapy ................................ 16
1.4 Aim of this work ....................................... 17
2 Material and Methods ........................................ 18
2.1 Restriction digestion .................................. 18
2.2 Gel electrophoresis and gel extraction ................. 18
2.3 Ligation reaction ...................................... 18
2.4 Propagation of plasmids in bacteria .................... 18
2.5 Purification of plasmids ............................... 19
2.6 Cell culture ........................................... 19
2.7 Construction of the TagLuc fusion gene ................. 19
2.8 Plasmids for analysis of transformation and
luciferase activity of the TagLuc fusion protein ....... 20
2.9 Analysis of transformation and luciferase activity of
the TagLuc fusion protein .............................. 20
2.10 Western blot analysis .................................. 20
2.11 Plasmids for generation of transgenic mice ............. 21
2.12 Analysis of Cre-mediated recombination of
LoxP-TagLuc-pA and LoxP-TagLuc plasmids ................ 21
2.13 Mice ................................................... 21
2.14 PCR .................................................... 22
2.15 Bioluminescent imaging ................................. 22
2.16 Histology and immunohistochemistry ..................... 23
2.17 In vivo cytotoxicity assay ............................. 23
2.18 ELISA .................................................. 24
2.19 Adoptive T cell transfer ............................... 24
3 Results ..................................................... 25
3.1 Design and in vitro analysis of the TagLuc fusion
gene ................................................... 25
3.1.1 Western blot analysis of the TagLuc fusion
protein ......................................... 26
3.1.2 Oncogenic and luciferase activity of the
TagLuc protein .................................. 28
3.2 Development of conditional TagLuc transgenic mice ...... 30
3.2.1 Generation of LoxP-TagLuc-pA transgenic mice
and in vivo expression of TagLuc ................ 30
3.2.2 CTL response towards Tag in LoxP-TagLuc-pA
transgenic mice ................................. 32
3.2.3 Generation of LoxP-TagLuc transgenic mice ....... 34
3.2.4 CTL response towards Tag in LoxP-TagLuc
transgenic mice ................................. 38
3.2.5 Cre-independent TagLuc gene expression in
individual organs from LoxP-TagLuc-pA and
LoxP-TagLuc transgenic mice ..................... 39
3.2.6 Analyzing in vitro expression of
LoxP-TagLuc-pA and LoxP-TagLuc constructs by
transient transfection .......................... 42
3.3 Tumor development in LoxP-TagLuc mice .................. 43
3.3.1 LoxP-TagLuc mice did not develop sporadic
tumors .......................................... 43
3.3.2 Detection of hepatocellular carcinoma in
LoxP-TagLuc transgenic mice crossed to liver-
specific Alb-Cre-trangenic mice ................. 45
3.3.3 Ex vivo analysis of the liver tumor from
LoxP-TagLuc x Alb-Cre double transgenic mice .... 47
3.4 Evaluating the therapeutic potential of adoptively-
transferred Tag-specific T cells into LoxP-TagLuc and
LoxP-TagLuc-pA mice ..................................... 48
4 Discussion .................................................. 50
5 References .................................................. 54
6 Summary ..................................................... 54
7 Zusammenfassung ............................................. 64
8 Acknowledgements ............................................ 65
9 Appendix .................................................... 66
Plasmid maps ................................................ 66
LoxP-TagLuc-pA .............................................. 66
LoxP-TagLuc ................................................. 67
pCAG-Tag .................................................... 68
pCAG-TagLuc ................................................. 69
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