A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Heidelberg
[2017]
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | V, 105 Seiten Illustrationen, Diagramme 30 cm |
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MARC
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| 100 | 1 | |a Eckert, Elias Simon Peter |e Verfasser |0 (DE-588)1150713860 |4 aut | |
| 245 | 1 | 0 | |a A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |c presented by Elias Simon Peter Eckert, M.Sc. |
| 264 | 1 | |a Heidelberg |c [2017] | |
| 300 | |a V, 105 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto-Carola University of Heidelberg |d 2017 | ||
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Record in the Search Index
| _version_ | 1804178304662503424 |
|---|---|
| adam_text | T
ABLE
OF
C
ONTENTS
1.
INTRODUCTION...........................................................................................................................................1
1.1 THE HEMATOPOIETIC SYSTEM - MAKING BLOOD
............................................................
.
..................1
1.1.1 ADULT
HEMATOPOIESIS................................................................................................................
1
1.1.2 HEMATOPOIETIC STEM
CELLS........................................................................................................
3
1.2 MICRO-RNAS * CENTRAL BALANCERS OF GENE
EXPRESSION...............................................................
11
1.2.1 BIOGENESIS OF MIRNAS................ 12
1.2.2 MIRNAS INTEGRATE CELLULAR
SYSTEMS.......................................................................................13
1.2.3 MIRNA-REGULATION OF NORMAL AND MALIGNANT
HEMATOPOIESIS.............................................15
1.3 RETROVIRAL INTEGRATION SITE ANALYSIS FOR THE IDENTIFICATION OF
CELLULAR REGULATORS
.....................
17
1.3.1 GENERAL PRINCIPLES OF RETROVIRAL
INTEGRATION.........................................................................
17
1.3.2 RETROVIRAL
INSERTIONAL-MUTAGENESIS........................................................................................
19
1.3.3 INTEGRATION SITE ANALYSIS IN GENE-THERAPY
TRIALS......................................................................20
1.3.4 GENE THERAPY OF WISKOTT-ALDRICH
SYNDROME.......................................................................22
2. SPECIFIC
AIMS.......................................................................................................................................
24
3.
RESULTS.................................................................................................................................................25
3.1 IDENTIFICATION OF POTENTIAL NOVEL HEMATOPOIETIC REGULATORY GENES BY
GAMMARETROVIRAL
INTEGRATION SITE
ANALYSIS..............................................................................................................25
3.1.1 ANALYSIS OF COMMON VECTOR INTEGRATION SITES FROM A WAS GENE
THERAPY STUDY
................
25
3.1.2 EXPRESSION OF CANDIDATE MIRNAS IN HEMATOPOIETIC
POPULATIONS......................................32
3.2 ESTABLISHMENT OF A LENTIVIRAL-BASED SCREENING PLATFORM FOR THE
FUNCTIONAL VALIDATION OF
HEMATOPOIETIC
REGULATORS...........................................................................................................33
3.2.1 STABLE MIRNA OVEREXPRESSION MEDIATED BY LENTIVIRAL
VECTORS...........................................33
3.2.2 MOLECULAR BARCODING CASSETTE IS SUITED FOR MULTIPLEXED
HIGH-THROUGHPUT SEQUENCING
......
35
3.2.3 HOMOGENEOUS LIBRARY REPRESENTATION IN TRANSDUCED LSK
CELLS..........................................36
3.3 IN VITRO AND IN VIVO IDENTIFICATION OF HEMATOPOIETIC REGULATORS
.................................................
37
3.3.1 SERIAL COLONY-FORMING UNIT ASSAYS REVEALS DIFFERENTIAL REPLATING
EFFICIENCIES DEPENDING ON
MIRNA OVEREXPRESSION
.............
37
3.3.2 BARCODED MIRNA OVEREXPRESSING SCREEN IN HEMATOPOIETIC STEM AND
PROGENITOR CELLS
REVEALS HEMATOPOIETIC REGULATORS IN VIVO
...............................................................................
39
3.4 OVEREXPRESSION OF MIR-LOA AND MIR-335 ALTERS HEMATOPOIETIC STEM AND
PROGENITOR OUTPUT
IN VITRO AND IN
VIVO........................................................................................................................43
3.4.1 SINGLE CANDIDATE SERIAL CPU ASSAYS CONFIRM IN VITRO SCREEN
..................................................
43
3.4.2 STEM AND PROGENITOR CELLS OVEREXPRESSING MIR-LOA OR MIR-335 SHOW
ALTERED LYMPHOCYTE
PRODUCTION..............................................................................................................................
43
3.4.3 OVEREXPRESSION OF MIR-LOA OR MIR-335 IN STEM AND PROGENITOR CELLS
INFLUENCES
PROGENITOR CELL
FRACTIONS........................................................................................................
45
3.4.4 OVEREXPRESSION OF MIR-LOA AND MIR-335 ALTER LSK-CELL GENE
EXPRESSION OF
LYMPHOPOIESIS RELATED PATHWAYS...................
.......................................................................
46
3.4.5 RISC IMMUNOPRECIPITATION INDICATES POTENTIAL MIR-LOA TARGETS
..........................................
48
4.
DISCUSSION............................................................................................................................................50
4.1 THE WAS DERIVED GAMMARETROVIRAL CD34+ INTEGROME MARKS
MIRNA-REGULATORS BUT NOT
PROTO-ONCOGENES.........................................................................................................................50
4.1.1 GAMMARETROVIRAL INTEGRATION SITES FROM THE WAS GENE THERAPY TRIAL
MARK REGULATORY
ELEMENTS IN LONG-TERM REPOPULATING
CELLS...............................................................................51
4.1.2 ONLY A FEW EXCEPTIONALLY LARGE CIS BUT NOT ALL INTEGRATION
HOTSPOTS MARK PROTO
ONCOGENES
............................
52
4.2 IMPROVEMENTS IN FUNCTIONAL, BARCODE-BASED SCREENING METHODS FOR
HEMATOPOIETIC
REGULATORS
..............
54
4.2.1 MIRNA OVEREXPRESSION SCREEN BEST RESEMBLES POTENTIAL RETROVIRAL
MEDIATED GAIN-OF-
FUNCTION
....
:..............................................................................................................................54
4.2.2 A FOCUSED, MEDIUM-THROUGHPUT SCREENING APPROACH IS MOST SUITABLE
TO DETECT CHANGES IN
HEMATOPOIETIC LINEAGES IN
VIVO................................................................................................55
4.3 THE ROLE OF MIR-LOA AND MIR-335 IN
HEMATOPOIESIS...............................................................60
4.3.1 MIR-335 INFLUENCES T- AND B-LYMPHOPOIESIS
...............
60
4.3.2 MIR-LOA LEADS TO INCREASED T CELL NUMBERS AND PROGENITOR CELLS
.........................
61
4.4 CONCLUSIONS AND
PERSPECTIVES....................................................................................................64
5. MATERIAL AND M
ETHODS................................................................................................................
.
......
65
5.1
MATERIALS......................................................................................................................
65
5.1.1 TECHNICAL EQUIPMENT AND
DEVICES.........................................................................................
65
5.1.2 COMMERCIAL
KITS.....................................................................................................................
66
5.1.3
REAGENTS..................................................................................................................................66
5.1.4 CONSUMABLES.
68
5.1.5
PLASMIDS...................................................................................................................................68
5.1.6
ANTIBODIES...............................................................................................................................68
5.1.7
OLIGONUCLEOTIDES.....................................................................................................................
71
5.1.8 MOUSE
STRAINS..........................................................................................................................74
5.1.9
SOFTWARE..................................................................................................................................
75
5.2 M
ETHODS.......................................................................................................................................
75
5.2.1 MOLECULAR
BIOLOGY...................................................................................................................75
5.2.2 CELL
BIOLOGY.............................................................................................................................78
5.2.3 ANIMAL
EXPERIMENTS...............................................................................................................
81
5.2.4 COMPUTATIONAL
ANALYSIS..........................................................................................................82
5.2.5 STATISTICAL ANALYSIS
..........................
83
6.
REFERENCES............................................................................................................................................84
7.
ACKNOWLEDGEMENTS............................................................................................................................102
7.1
CONTRIBUTIONS............................................................................................................................102
7.2 PERSONAL
ACKNOWLEDGEMENTS....................................................................................................102
8.
ABBREVIATIONS....................................................................................................................................
104
|
| any_adam_object | 1 |
| author | Eckert, Elias Simon Peter |
| author_GND | (DE-588)1150713860 |
| author_facet | Eckert, Elias Simon Peter |
| author_role | aut |
| author_sort | Eckert, Elias Simon Peter |
| author_variant | e s p e esp espe |
| building | Verbundindex |
| bvnumber | BV044792409 |
| ctrlnum | (OCoLC)1135418680 (DE-599)DNB1150879505 |
| discipline | Biologie |
| format | Thesis Book |
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| id | DE-604.BV044792409 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T08:01:51Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-030187585 |
| oclc_num | 1135418680 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | V, 105 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2017 |
| publishDateSearch | 2017 |
| publishDateSort | 2017 |
| record_format | marc |
| spelling | Eckert, Elias Simon Peter Verfasser (DE-588)1150713860 aut A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis presented by Elias Simon Peter Eckert, M.Sc. Heidelberg [2017] V, 105 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto-Carola University of Heidelberg 2017 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1150879505/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030187585&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Eckert, Elias Simon Peter A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |
| subject_GND | (DE-588)4113937-9 |
| title | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |
| title_auth | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |
| title_exact_search | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |
| title_full | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis presented by Elias Simon Peter Eckert, M.Sc. |
| title_fullStr | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis presented by Elias Simon Peter Eckert, M.Sc. |
| title_full_unstemmed | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis presented by Elias Simon Peter Eckert, M.Sc. |
| title_short | A pooled lentiviral miRNA overexpression screen in hematopoietic stem and progenitor cells reveals miRNA-10a and miRNA-335 as regulators of lymphopoiesis |
| title_sort | a pooled lentiviral mirna overexpression screen in hematopoietic stem and progenitor cells reveals mirna 10a and mirna 335 as regulators of lymphopoiesis |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1150879505/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030187585&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT eckerteliassimonpeter apooledlentiviralmirnaoverexpressionscreeninhematopoieticstemandprogenitorcellsrevealsmirna10aandmirna335asregulatorsoflymphopoiesis |
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