Generation and characterization of recombinant cell penetrating peptides for siRNA delivery:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XI, 154 S. Ill., graph. Darst. 30 cm |
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| adam_text | IMAGE 1
CONTENTS
ABBREVIATIONS V
ABSTRACT VIII
ABSTRACT (DEUTSCH) X
CHAPTER I THE POTENTIAL O F CELL PENETRATING PEPTIDES AS A VEHICLE FOR
THE DELIVERY OF
SIRNA: AN OVERVIEW AND WORKING HYPOTHESIS 1
1 CELL-PENETRATING PEPTIDE (CPP) 2
2 RNA INTERFERENCE (RNAI) 4
3 CPP BASED SIRNA DELIVERY 6
3.1 CPP MEDIATED SIRNA DELIVERY 6
3.2 CPP AND OTHER VECTOR CONJUGATED SIRNA DELIVERY 8
3.2. 1 LIPOSOME MODIFIED CPP 8
3.2.2 COMBINATION O F CPP WITH POLYMER 11
3.2.3 COMBINATION O F CPP WITH CATIONIC PEPTIDE 12
3.2.4 NANOPARTICLES MODIFIED WITH CPP 14
3.2.5 BINDING O F CPP WITH THE VIRAL CARRIER 17
3.3 CALCIUM AND CPP 18
4 WORKING HYPOTHESIS AND THE DESIGN O F PROPOSED RECOMBINANT PROTEINS
FOR SIRNA DELIVERY
20
CHAPTER II GENERATION O F RECOMBINANT PROTEINS FOR SIRNA DELIVERY:
DESIGN,
BIOINFORMATIC ANALYSIS, CLONING AND PROTEIN GENERATION 23
1 INTRODUCTION 24
2 MATERIALS 25
2.1 CHEMICALS AND INSTRUMENTS 25
2.1.1 CHEMICALS 25
2.1.2 EXPENDABLES 25
2.2 NUCLEAR ACIDS 25
2.3 BACTERIA STRAINS 26
2.4 MOLECULAR WEIGHT STANDARDS 26
2.5 ENZYMES 26
2.6 ANTIBODIES 27
I
HTTP://D-NB.INFO/1032229446
IMAGE 2
2.7 BUFFERS, SOLUTIONS AND SUPPLEMENTS 27
2.8 COMMERCIALLY AVAILABLE KITS 29
2.9 INSTRUMENTS AND EQUIPMENT 30
3 METHODS 31
3.1 PREPARATION O F MINI PLASMID 31
3.2 DNA CONCENTRATION DETERMINATION 31
3.3 RESTRICTION DIGESTION ..31
3.4 DNA AGAROSE ELECTROPHORESIS AND RECOVERY 32
3.5 T4 DNA LIGATION 32
3.6 LB AGAROSE PLATE PREPARATION 33
3.7 TRANSFORMATION O F E.COLI 33
3.8 SEQUENCING 33
3.9 BIOINFORMATICS ANALYSIS 34
3.10 PROTEIN EXPRESSION OPTIMIZATION 34
3.11 SDSPAGE 34
3.12 WESTERN BLOT 35
3.13 PROTEIN ISOLATION 36
3.14 DETERMINATION O F PROTEIN CONCENTRATION 39
4 RESULTS 40
4.1 CHARACTERIZATION O F THE SIRNA DELIVERY VECTOR 40
4.2 STRUCTURE PREDICTION O F THESE PROTEINS 41
4.3 GENERATION O F EXPRESSION PLASMIDS 43
4.4 RESTRICTION DIGESTION DETERMINES THE COLON AND SEQUENCE ANALYSIS 45
4.5 OPTIMIZATION O F THE CONDITION FOR THE EXPRESSION O F RECOMBINANT
PROTEINS IN BACTERIA
47
4.6 PROTEIN PURIFICATION AND DETERMINATION 53
5 DISCUSSION 56
6 SUMMARY 62
CHAPTER III EFFICACY OF GENERATED RECOMBINANT PROTEINS FOR THE SIRNA
DELIVERY: IN VITRO
SETTINGS 63
1 INTRODUCTION 64
2 METHODS 67
2.1 PREPARATION O F PROTEIN-SIRNA CONDENSED WITH CACFE 67
II
IMAGE 3
2.2 PREPARATION O F PROTEIN-SIRNA 67
2.3 AGAROSE GEL RETARDATION 67
2.4 CELL UP-TAKING FLUORESCENCE MICROSCOPY 68
2.5 CELL CULTURE 68
2.8 GENE KNOCKED DOWN IN VITRO 70
2.9 FLOW CYTOMETRY ANALYSIS (FACS) 70
2.10 STATISTICAL ANALYSIS 71
3 RESULTS 72
3.1 DETECTION O F PROTEIN-SIRNA INTERACTIONS BY GEL RETARDATION ASSAYS
72
3.2 MICROSCOPICALLY ANALYSIS O F SIRNA TRANSFECTION 74
3.3 EXOGENOUS GFP KNOCKED DOWN IN VITRO 77
3.3.1 GENERATION O F HELA CELL LINE STABLY EXPRESSING GFP 77
3.3.2 EXOGENOUS GFP KNOCKED DOWN IN GFP-EXPRESSION HELA CELLS 78
3.4 ENDOGENOUS GENE KNOCKED DOWN IN HELA CELL ...81
3.5 CELL VIABILITY 83
4 DISCUSSION 85
5 SUMMARY 89
CHAPTER IV CHARACTERIZATION O F A RECOMBINANT PROTEIN FOR IN VIVO SIRNA
DELIVERY 90
1 INTRODUCTION 91
2 METHODS 92
2.1 ISOLATION AND CULTURE O F PRIMARY CELLS 92
2.2 CELL STAINING 92
2.3 CONSTRUCTION O F HEART TRANSPLANTATION MODEL 92
2.4 HISTOLOGICAL ANALYSIS 93
2.5 REAL TIME PCR 93
3 RESULTS 95
3.1 TRANSFER O F PROTEIN 9- SIRNA COMPLEXES TO CELLS 95
3.2 PROTEIN 9 TRANSFERRING SIRNA TO CELLS WITH DIFFERENT HOMING
PROPERTIES 98
3.3 OPTIMIZATION O F THE EFFICIENCY O F PROTEIN 9 FACILITATED SIRNA
DELIVERY 99
3.3.1 EFFECT O F TRANSFECTION TIME DURATION ON PROTEIN 9-BASED FAM-SIRNA
DELIVERY 99
3.3.2 EFFECT O F TEMPERATION ON THE PROTEIN 9 DELIVERY O F SIRNA INTO
CELLS 101
HI
IMAGE 4
3.4 GENE SILENCING CAUSED BY PROTEIN 9-SIGFP COMPLEX ON GFP EXPRESSION
HELA CELLS
102
3.5 GENE SILENCING ON ENDOGENOUS GENE BY PROTEIN 9 - ANTI GAPDH SIRNA
103
3.6 CALCIUM ENHANCES THE EFFICIENCY O F PROTEIN 9-BASED SIRNA DELIVERY
104
3.6.1 CALCIUM CONDENSED PROTEIN-SIRNA COMPLEXES OFFER AN IMPROVED
TRANSFECTION EFFICACY 104
3.6.2 INVESTIGATION O F SILENCING ENDOGENOUS GENE GAPDH USING CALCIUM
CONDENSATION 108
3.6.3 THE MECHANISM O F THE CONDENSING EFFECT IN ENHANCING SIRNA
DELIVERY
EFFICIENCY 109
3.8 APPLICATION O F PROTEIN 9 IN VIVO I L L
3.8.1 CONSTRUCTION O F MOUSE HEART TRANSPLANTATION MODEL I L L
3.8.2 DISTRIBUTION O F PROTEIN-SIRNA COMPLEXES IN THE MOUSE HEART 113
3.8.3 PROTEIN 9 APPLIED IN THE MOUSE HEART TRANSPLANT 115
4 DISCUSSION 116
5 SUMMARY 121
REFERENCES 122
ACKNOWLEDGEMENTS 139
APPENDICES 140
1 MAP O F PET 28B+ 140
2 MAP OFPCDNA3.1 141
3 SEQUENCE SYNTHESIS 142
4 SEQUENCE O F PROTEIN 144
5 CURRICULUM VITAE 146
6 DECLARATION (UP ARM) 148
7 THE GEFAHRENSYMBOLE, H&P SATZE 149
IV
|
| any_adam_object | 1 |
| author | Li, Hua 1984- |
| author_GND | (DE-588)103209950X |
| author_facet | Li, Hua 1984- |
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| building | Verbundindex |
| bvnumber | BV041369553 |
| ctrlnum | (OCoLC)843424428 (DE-599)DNB1032229446 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| language | English |
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| physical | XI, 154 S. Ill., graph. Darst. 30 cm |
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| spelling | Li, Hua 1984- Verfasser (DE-588)103209950X aut Generation and characterization of recombinant cell penetrating peptides for siRNA delivery vorgelegt von Hua Li 2012 XI, 154 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Hamburg, Univ., Diss., 2012 RNS-Interferenz (DE-588)4802706-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content RNS-Interferenz (DE-588)4802706-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026817725&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Li, Hua 1984- Generation and characterization of recombinant cell penetrating peptides for siRNA delivery RNS-Interferenz (DE-588)4802706-6 gnd |
| subject_GND | (DE-588)4802706-6 (DE-588)4113937-9 |
| title | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery |
| title_auth | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery |
| title_exact_search | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery |
| title_full | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery vorgelegt von Hua Li |
| title_fullStr | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery vorgelegt von Hua Li |
| title_full_unstemmed | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery vorgelegt von Hua Li |
| title_short | Generation and characterization of recombinant cell penetrating peptides for siRNA delivery |
| title_sort | generation and characterization of recombinant cell penetrating peptides for sirna delivery |
| topic | RNS-Interferenz (DE-588)4802706-6 gnd |
| topic_facet | RNS-Interferenz Hochschulschrift |
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