Capsid-engineering overcomes barriers toward endothelial cell transduction:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Köln
2015
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| Beschreibung: | V, 98 Seiten Illustrationen, Diagramme 21 cm |
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Datensatz im Suchindex
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| adam_text | Titel: Capsid-engineering overcomes barriers toward endothelial cell transduction
Autor: Zhang, Li-Ang
Jahr: 2015
Table of contents
SUMMARY....................................1
ZUSAMMENFASSUNG.............................2
INTRODUCTION........................................................................................................3
2.1 Chemicals, solutions and enzymes....................................................................19
2.2 Reagent setup.....................................................................................................20
2.3 Standard kits......................................................................................................21
2.4 Plasmids..............................................................................................................22
2.5 Primers................................................................................................................23
2.6 Single-stranded oligonucleotides...................................................................24
2.7 Antibodies............................................................................................................24
2.8 Bacteria strain...................................................................................................25
2.9 Eukaryotic cells...............................................................................................25
2 10 Equipments and disposables..............................................................................25
2 11 Data treating son ware.....................................................................................26
3 METHODS...............................................................................................................27
3.1 Bacteria...............................................................................................................27
3.1.1 Cultivation of bacteria...................................................................................................27
3.1.2 Preparation of chemically competent bacteria.........................................................27
3.1.3 Transformation of bacteria...........................................................................................27
3.1.4 DNA expansion in bacteria..........................................................................................28
3.2 Modification, purification and quantification of nucleic acid........................28
3.2.1 Oligonucleotide hybridization......................................................................................28
3.2.2 Production ofpLG construct........................................................................................28
3.2.3 Production of pLG backbone.......................................................................................29
3.2.4 Production of AA V sub-library plasmid pool.............................................................29
3.2.5 DNA purification by gel electrophoresis....................................................................30
3.2.6 DNA extraction from cells............................................................................................31
3.2.7 RNA extraction from cells............................................................................................31
3.2.8 Measurement of nucleic acid concentration.............................................................31
3.3 Polymerase Chain Reaction (PGR)....................................................................31
3.3.1 PCR for cloning.............................................................................................................31
3.3.2 PCR for Sanger sequencing........................................................................................33
3.3.3 Real-time PCR (qPCR)................................................................................................34
3.3.4 Reverse transcriptase PCR (RT-PCRj......................................................................36
3.4 NEXT GENERATION SEQUENCING.............................................................................37
3.4.1 Preparation of amplicon...............................................................................................37
3.4.2 Emulsion PCR...............................................................................................................37
3.4.3 454-pyrosequencing and analysis of sequences.....................................................37
3.5 Protein................................................................................................................37
3.5.1 Acetone precipitation of protein fractions..................................................................37
3.5.2 Western Blot..................................................................................................................38
3.6 Eukaryotic cell culture....................................................................................39
3.6.1 Cultivation of cells.........................................................................................................35
3.6.2 Trypsin and accutase treatment.................................................................................40
3.6.3 Counting, seeding and passaging..............................................................................40
3.6.4 Freezing and thawing cells..........................................................................................40
3.6.5 Cell fractionation...........................................................................................................40
3.6.6 Induction of quiescent HUVEC.......................................................41
3.7 AAV library.........................................................................................................41
3.7.1 AAV library production and purification.....................................................................41
3.7.1.1 AAV library packaging.............................................................................................41
3.7.1.2 lodixanol gradient purification................................................................................42
3.7.1.3 Vector titration........................................................................................................42
3.7.1.4 Coupling of pheno- and geno-type of mutants.......................................................43
3.7.2 AAV library screening...................................................................................................43
3.8 Cell transduction assay by capsid-modified MV vectors..............................44
3.8.1 AAV vector production.................................................................................................44
3.8.2 Transduction of HUVEC by capsid-modified AAV vectors.....................................45
3.8.3 Cell transduction assay................................................................................................45
3.8.3.1 Flow cytometry........................................................................................................45
3.8.3.2 Benzonase protection assay....................................................................................46
3.8.3.3 Quantification of vector entry efficiency.................................................................46
3.9 Statistical analysis............................................................................................46
4 RESULTS.................................................................................................................47
4.1 Selection of EC variants...................................................................................47
4.1.1 Characterization of cell surface receptors.................................................................47
4.1.2 Using the targeting technology to understand barriers of EC transduction.........49
4.1.3 Selection criteria............................................................................................................49
4.1.4 Selection procedure......................................................................................................49
4.2 Characterization of selected variants............................................................56
4.2.1 AAV capsid-modified vectors transduce HUVEC with higher efficiency..............56
4.2.2 AAV capsid-modified vectors enter cells more efficient thanAAV2......................57
4.2.3 AAV capsid-modified vectors outperform AAV2 in number of particles that
accumate in the perinuclear area...............................................................................58
4.2.4 AAV capsid-modified vectors differ in the onset of transgene expression...........59
4.2.5 The benzonase protection assay does not reveal any difference in uncoating
efficiency........................................................................................................................60
4.2.6 Single-strand conversion is a post-entry barrier that hampers EC transduction. 61
4.2.7 AAV-V is inhibited by heparin like AA V2 in EC transduction.................................62
4.2.8 Empty capsid of AA V-V and AA V2 do not enter the nuclear area........................63
4.2.9 AAV-V is superior to the variants identified in previous studies in EC
transduction...................................................................................................................63
4.3 Characterization of selected variants in preliminary study for in vivo
TESTS....................................................................................................................64
5 DISCUSSION.............................................................................................................
5.1 AAVLIBRARY SCREENING WITH NGS.......................................................................69
5.2 Cell receptor interaction.................................................................................71
5.3 Cell entry barriers............................................................................................73
5.4 Summary and perspec tivesofAAV- V s applica tion..........................................75
6 REFERENCE...................................................77
S ERKLARUNG.......................................................98
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| bvnumber | BV043858849 |
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| dewey-ones | 572 - Biochemistry |
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| dewey-tens | 570 - Biology |
| discipline | Biologie Medizin |
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| indexdate | 2024-07-10T07:36:56Z |
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| language | English |
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| spelling | Zhang, Li-Ang 1982- Verfasser (DE-588)1106258037 aut Capsid-engineering overcomes barriers toward endothelial cell transduction vorgelegt von Li-Ang Zhang Köln 2015 V, 98 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2016 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1110917694/04 Inhaltsverzeichnis HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029269003&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Zhang, Li-Ang 1982- Capsid-engineering overcomes barriers toward endothelial cell transduction |
| subject_GND | (DE-588)4113937-9 |
| title | Capsid-engineering overcomes barriers toward endothelial cell transduction |
| title_auth | Capsid-engineering overcomes barriers toward endothelial cell transduction |
| title_exact_search | Capsid-engineering overcomes barriers toward endothelial cell transduction |
| title_full | Capsid-engineering overcomes barriers toward endothelial cell transduction vorgelegt von Li-Ang Zhang |
| title_fullStr | Capsid-engineering overcomes barriers toward endothelial cell transduction vorgelegt von Li-Ang Zhang |
| title_full_unstemmed | Capsid-engineering overcomes barriers toward endothelial cell transduction vorgelegt von Li-Ang Zhang |
| title_short | Capsid-engineering overcomes barriers toward endothelial cell transduction |
| title_sort | capsid engineering overcomes barriers toward endothelial cell transduction |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1110917694/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029269003&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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