Targeting immunotoxins to activated T-lymphocytes:
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
2000
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Freiburg (Breisgau), Univ., Diss., 2001 (Nur beschränkt für den Austausch) |
| Physical Description: | 102, VIII Bl. Ill., graph. Darst. : 30 cm |
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|---|---|
| adam_text |
_I_
CONTENTS I
A INTRODUCTION 1
1 IN VIVO APPLICATION OF RECOMBINANT HEMATOPOIETIC CYTOKINES 1
2 APPLICATION OF SYNGENEIC AND ALLOGENEIC CYTOKINE GENE
TRANSFECTED FIBROBLASTS 1
2.1 IRRADIATED CYTOKINE GENE TRANSDUCED SYNGENEIC CELLS DELIVER
BIOLOGICALLY ACTIVE LEVELS OF CYTOKINES IN VIVO 1
2.2 A SECOND TRANSPLANTATION OF CYTOKINE GENE TRANSFECTED ALLOGENEIC
FIBROBLASTS DOES NOT ACHIEVE THERAPEUTIC EFFECTS 2
3 MECHANISMS OF ALLOREJECTION 3
4 IMMUNOSUPPRESSION OF ALLOREACTIVITY 5
4.1 UNSPECIFIC T CELL TARGETED IMMUNOSUPPRESSION 5
4.2 SPECIFIC IMMUNOSUPPRESSION BY TARGETING IL2 RECEPTOR POSITIVE
CELLS 6
4.2.1 IL2-PSEUDOMONAS EXOTOXIN IMMUNOTOXIN 7
4.2.2 ANGIOGENIN-IL2 IMMUNOTOXIN 10
B STUDY OBJECTIVES 12
C MATERIALS AND METHODS 17
1
1.1
MATERIALS
REAGENTS AND THEIR SOURCE
17
17
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/961942851
_N _
1.2 SOLUTIONS AND BUFFERS 19
1.3 SUPPLEMENTED MEDIA 21
1.4 CELL LINES 21
1.5 ANIMALS 22
2 MOLECULAR BIOLOGY AND PROTEIN CHEMISTRY 22
2.1 DNA PREPARATION 22
2.1.1 CONCENTRATION OF DNA BY ETHANOL, PRECIPITATION 22
2.1.2 SPECTROPHOTOMETRIC DETERMINATION OF DNA CONCENTRATION 22
2.2 AGAROSE GEL ELECTROPHORESIS 23
2.3 DNA EXTRACTION FROM LOW MELTING POINT GEL 23
2.4 ENZYMATIC MANIPULATION OF DNA 24
2.4.1 DIGESTION OF DNA WITH RESTRICTION ENDONUCLEASES 24
2.4.2 DEPHOSPHORYLATION OF LINEAR DNA WITH PHOSPHATASE 24
2.4.3 LIGATION OF DNA FRAGMENTS IN GEL SLICES 24
2.5 TRANSFORMATION IN E. COLI 25
2.5.1 TRANSFORMATION BY ELECTROPORATION 25
2.5.2 BLUE-WHITE SELECTION FOR SCREENING RECOMBINANT CLONES 25
2.6 ISOLATION OF PLASMID DNA FROM BACTERIA 26
2.6.1 PLASMID PREPARATION IN SMALL SCALE 26
2.6.2 PLASMID PREPARATION IN LARGE SCALE 26
2.7 SEQUENCE ANALYSIS 27
2.8 POLYMERASE CHAIN REACTION AND CONSTRUCTION OF EUKARYOTIC
EXPRESSION VECTOR 27
2.8.1 MRNA EXTRACTION FROM NS3 CELLS 27
2.8.2 FIRST STRAND CDNA SYNTHESIS 28
2.8.3 HUMAN ANGIOGENIN SPECIFIC CDNA SYNTHESIS BY PCR(L) 29
2.8.4 SY NTHESI S OF H ANG+LINKER BY PCR(2) 29
2.8.5 SYNTHESI S OF HIL2+LINKER BY PCR 29
2.8.6 FUSION OF ANGIL2 IN PBLUESCRIPT 29
2.8.7 CONSTRUCTION OF EXPRESSION VECTOR 30
2.8.8 SUMMARY OF CLONING PROCEDURE 30
2.9 WESTERN BLOT 32
_M_
2.9.1 INTRODUCTION 32
2.9.2 CELL LYSIS 32
2.9.3 DETERMINATION OF PROTEIN CONCENTRATION 33
2.9.4 SDS-PAGE ELECTROPHORESIS 33
2.9.5 PROTEIN TRANSFER (BLOT) 34
2.9.6 IMMUNOPROBING AND VISUAL DETECTION OF PROTEINS 35
2.10 NORTHERN BLOT 35
2.10.1 AGAROSE/FORMALDEHYDE GEL ELECTROPHORESIS 35
2.10.2 TRANFER OF RNA FROM GEL TO MEMBRANE 36
2.10.3 IMMOBILIZATION OF RNA AND PREPARATION OF PROBES 36
2.10.4 HYBRIDIZATION 37
2.10.5 AUTORADIOGRAPHY 37
3 CELL BIOLOGY ~ 37
3.1 CELL CULTURE 37
3.1.1 DETERMINATION OF VIABLE CELLS (TRYPAN BLUE EXCLUSION TEST FOR CELL
VIABILITY) 37
3.1.2 MAINTENANCE OF THE CULTURE-CELL LINES 38
3.1.3 CELL FREEZING 38
3.1.4 CELL THAWING 38
3.1.5 ALLOGENIC SPECIFIC LYMPHOCYTE EXPANSION FOR BIOASSAYS 39
3.2 TRANSFECTION IN FIBROBLASTS 39
3.3 DILUTION CLONING AND SELECTION 39
3.4 DETERMINATION OF CYTOKINE CONCENTRATION IN CULTURE SUPERNATANTS 40
3 .4.1 SUPERNATANT COLLECTION 40
3.4.2 ELISA 40
3.5 SCREENING IL2 RECEPTOR POSITIVE CELL LINES, 41
3.6 BIOASSAY 41
4 IN VIVO EXPERIMENTS 42
4.1 CHEMOTHERAPY AND TRANSPLANTATION 42
4.2 BLOOD COLLECTION AND MEASUREMENT OF PERIPHERAL BLOOD CELLS 44
IV
D
RESULTS
45
1 CLONING, EXPRESSION AND IN VITRO ANALYSIS OF A T CELL TARGETED
IMMUNOTOXIN DERIVED FROM PSEUDOMONAS EXOTOXIN 45
1.1 CONSTRUCTION OF THE EUKARYOTIC EXPRESSION VECTOR FOR IL2PE
USING A POLYA CONTAINING INTRON 45
1.1.1 CLONING STRATEGY WITH AN INTRON CONTAINING POLYA 45
1.1.2 TRANSFECTION OF BALB3T3 FIBROBLASTS AND ANALYSIS OF EXPRESSION 48
1.1.3 IMMUNOBLOTTING OF PROTEIN EXPRESSED BY TRANSFECTED BALB3T3
CLONES 49
1.1.4 NORTHERN BLOT ANALYSIS OF TRANSCRIPTS 50
1.1.5 SCREENING IL2 RECEPTOR POSITIVE CELL LINES 52
1.1.6 XTT ASSAY AND
3H THYMIDINE INCORPORATION ASSAY 53
1.2 CONSTRUCTION OF THE EUKARYOTIC EXPRESSION VECTOR FOR IL2PE
USING POLYA WITHOUT INTRON 53
2 CLONING, EXPRESSION, IN VITRO AND IN VIVO ANALYSIS OF A T CELL
TARGETED IMMUNOTOXIN DERIVED FROM HUMAN ANGIOGENIN 54
2.1 CONSTRUCTION OF AN EXPRESSION VECTOR FOR ANGIL2 EXPRESSION 54
2.1.1 CONSTRUCTION OF THE ANGIL2 FUSION GENE 55
2.1.2 CONSTRUCTION OF AN EUKARYOTIC EXPRESSION VECTOR OF ANGIL2 58
2.2 TRANSFECTION OF THE ANGIL2 EXPRESSION VECTOR IN MURINE
BALB3T3 FIBROBLASTS 60
2.3 SECRETION OF ANGIL2 FROM TRANSFECTED BALB3T3 60
2.4 BIOLOGICAL ACTIVITY OF ANGIL2 IN VITRO 62
2.4.1 SCREENING IL2 RECEPTOR POSITIVE CELL LINES' 62
2.4.2 XTT ASSAY 62
2.5 PROTECTIVE EFFECT ON HG-CSF SECRETING BALB3T3 IN THE
ALLOGENIC TRANSPLANTATION MOUSE MODEL 63
_Y_
E DISCUSSION 68
1 IL2PE IMMUNOTOXIN 68
1.1 CONSTRUCTION OF THE IL2PE EXPRESSION VECTOR 68
1.1.1 PROMOTER 69
1.1.2 NEO AND IRES 70
1.1.3 NTS 71
1.2 EXPRESSION AND SECRETION OF IL2PE IMMUNOTOXIN 72
2 ANGIL2 IMMUNOTOXIN 76
2.1 CONSTRUCTION OF THE ANGIL2 EXPRESSION VECTOR 76
2.2 EXPRESSION AND SECRETION OF THE ANGIL2 IMMUNOTOXIN 77
2.3 BIOLOGICAL EFFECTS OF ANGEL2 IN VITRO AND IN VIVO 78
2.4 INDUCTION OF PERIPHERAL TOLERANCE 82
2.5 PROSPECTIVES 83
F SUMMARY 85
G REFERENCES 86
\
ABBREVIATIONS VI
ACKNOWLEDGEMENTS
VIII |
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| spelling | Cao, Ou Verfasser aut Targeting immunotoxins to activated T-lymphocytes vorgelegt von Ou Cao 2000 102, VIII Bl. Ill., graph. Darst. : 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2001 (Nur beschränkt für den Austausch) Hochschulschrift (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009514643&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Cao, Ou Targeting immunotoxins to activated T-lymphocytes Hochschulschrift |
| subject_GND | (DE-588)4113937-9 |
| title | Targeting immunotoxins to activated T-lymphocytes |
| title_auth | Targeting immunotoxins to activated T-lymphocytes |
| title_exact_search | Targeting immunotoxins to activated T-lymphocytes |
| title_full | Targeting immunotoxins to activated T-lymphocytes vorgelegt von Ou Cao |
| title_fullStr | Targeting immunotoxins to activated T-lymphocytes vorgelegt von Ou Cao |
| title_full_unstemmed | Targeting immunotoxins to activated T-lymphocytes vorgelegt von Ou Cao |
| title_short | Targeting immunotoxins to activated T-lymphocytes |
| title_sort | targeting immunotoxins to activated t lymphocytes |
| topic | Hochschulschrift |
| topic_facet | Hochschulschrift |
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