Inhoudsopgave
Hoofdstuk 1: CRISPR/cas nuclease technologie en toepassingen.................................................. 5c
CRISPR cas...................................................................................................................................... 5
CRSPR cas technologie .................................................................................................................... 5
Dubbelstrengbeuk introduceren ....................................................................................................... 6
Cas9 ............................................................................................................................................... 6
Beperkingen transport CRISPR-cas ................................................................................................... 6
Verkrijgen functioneel cas9-sgRNA ribonucleoproteïne in de celkern .................................................. 7
CRISPR cas expressievector (knockout plasmide).............................................................................. 8
BPK764: NHEJ herstel ....................................................................................................................... 9
Transportmechanismen voor aanbreng CRIPR-cas in EU cellen.......................................................... 9
Alternatief gebruik van de CRISPR-cas technologie ...........................................................................10
RNA Editing .....................................................................................................................................11
Aan- en uitschakelen van CRISPR/cas-gene editing ..........................................................................11
Toepassingen CRISPR cas ...............................................................................................................11
Beperkingen van gebruik CRISPR cas ...............................................................................................11
Beperken aspecifieke effecten cas9 .................................................................................................12
Hoofdstuk 2: RNA isolatie ............................................................................................................ 12
2.1 Inleiding RNA – verschillende soorten RNAs ................................................................................12
2.2 RNA isolatiemethoden ...............................................................................................................12
2.2.1 Protocol RNA isolatie .......................................................................................................... 13
Hoofdstuk 3: In vitro cDNA synthese, RACE en miRNA PCR............................................................ 20
3.1 Aanmaak van DNA in vitro ..........................................................................................................20
3.1.1 Chemische DNA synthese ................................................................................................... 20
3.1.2 DNA printing: 3D printer....................................................................................................... 20
3.1.3 Aanmaak cDNA ................................................................................................................... 20
3.1.4 Aanmaak DNA via PCR ........................................................................................................ 22
3.2 Analyse van miRNA door RT-PCR ................................................................................................23
Hoofdstuk 4: Expressie analyse van RNA en DNA .......................................................................... 24
4.1 Kwantitatieve PCR - qPCR ..........................................................................................................24
4.1.1 Algemene detectie strategieën qPCR .................................................................................. 24
4.1.2 Theoretische aspecten qPCPR ............................................................................................ 26
4.1.3 Data-analyse qPCR ............................................................................................................ 27
4.2 Digitale druppel PCR ..................................................................................................................28
, 4.3 nCounter ...................................................................................................................................29
Hoofdstuk 5: Next generation sequentie analyse: DNA en RNA sequeneren ................................... 30
5.1 NGS ..........................................................................................................................................30
5.1.1 Aanmaak bibliotheek & belangrijkste methoden NGS (library prep)........................................ 30
5.1.2 Aanmaak RNA bibliotheek ................................................................................................... 31
5.1.3 Klonale expansie bibliotheek door polony of bridge PCR ....................................................... 33
5.1.4 Sequencing by synthesis ..................................................................................................... 34
5.1.5 NGS data analyse................................................................................................................ 34
5.2 Single cell NG sequencing ..........................................................................................................35
5.3 Spatial transcriptomics ..............................................................................................................36
Hoofdstuk 6: third generation sequencing .................................................................................... 36
6.1 Pacbio Sequel system ................................................................................................................36
6.3 Nanopore syteem ......................................................................................................................38
Hoofdstuk 7: mRNA transcript analyse ......................................................................................... 39
7.1 Inleiding ISH: CISH en FISH ........................................................................................................39
7.2 ViewRNA ISH Cell Assay.............................................................................................................40
7.3 DualRNAscope ISH-IHC .............................................................................................................40
7.4 MerFISH ....................................................................................................................................41
Hoofdstuk 8: RNA technologiën.................................................................................................... 42
8.1 Principe van antisense RNA/ RNA interference ............................................................................42
8.1.1 Aanmaak antisense en RNAi in het lab ................................................................................. 43
8.2 Circulair RNA .............................................................................................................................45
8.4 Long non-coding RNA ................................................................................................................45
Hoofdstuk 9: epigenetisch onderzoek........................................................................................... 46
9.1 Inleiding epigenetica ..................................................................................................................46
9.2 Epigenetische processen ...........................................................................................................47
9.2.1 DNA methylatie ................................................................................................................... 47
9.2.2 Histone modificatie enzymen .............................................................................................. 47
9.3 Technieken om epigenetische veranderingen en histone modificaties te bestuderen ....................47
9.3.1 Methylatiegevoelige restrictie enzymen ................................................................................ 47
9.3.2 Whole genome bisulfiet sequencen ..................................................................................... 48
9.2.3 Mass Array Sequenom epityper ...................................................................................... 49
9.2.4 ChipSeq immunoprecipitatie om histon modificaties te analyseren ................................. 50
9.3 Toepassingen: factoren van invloed op DNA methylatie veranderingen .........................................50
Hoofdstuk 10: Genexpressie in prokaryoten ................................................................................. 52
10.1 Inleiding en basisconcepten .....................................................................................................52
, 10.2 Opbouw prokaryote expressievector .........................................................................................53
10.3 Regulatie expressie met induceerbare bacteriële promotoren ....................................................54
10.4 Problemen en optimalisatie EU eiwitexpressie in PRO cel .........................................................56
10.5 Tags en fusiesystemen .............................................................................................................57
10.6 Courante expressievectoren ....................................................................................................60
10.6.1 pET ................................................................................................................................... 60
10.6.3 pTrcHis vector systeem ..................................................................................................... 61
10.6.3 pBAD/ His fusie-vector met polyHis-tagvectoren ................................................................ 61
10.6.4 pGEX fusie-vectoren met GST ............................................................................................ 61
10.7 Kloneringsmethoden ................................................................................................................62
10.7.1 SLIC ................................................................................................................................. 62
10.7.2 Gateway cloning ............................................................................................................... 62
10.8 Toepassingen ..........................................................................................................................63
Hoofdstuk 11: Genexpressie in Eukaryoten ................................................................................... 64
11.1 Expressie EU in insectencel ......................................................................................................64
11.2 Expressie EU in mammalia of zoogdiercellen .............................................................................64
11.3 Types virale vectoren ...............................................................................................................66
11.3.1 Adenovirus à ................................................................................................................... 66
11.3.2 Adeno-associated virus ..................................................................................................... 68
11.3.3 Retrovirus ......................................................................................................................... 69
11.3.4 Lentivirus .......................................................................................................................... 70
Hoofdstuk 12: functie analyse technieken .................................................................................... 73
12.1 Analyse DNA regulator gebieden ...............................................................................................73
12.1.1 DNAse 1 footprinting ......................................................................................................... 73
12.1.2 Elektroforetische mobiliteit shift assay .............................................................................. 74
12.1.3 ChIP chromatine immunoprecipitatie ................................................................................ 74
12.2 Reporter genen en deletie analyse ............................................................................................75
12.3 Analyse naar de interactie tussen EW .......................................................................................76
Hoofdstuk 13: Genetische modificatie van dieren ......................................................................... 76
13.1 Genetische manipulatie in dieren .............................................................................................76
13.2 Zebravis: model voor onderzoek naar humane ziekten ...............................................................76
13.3 Meest gebruikte methoden om zebravis gen functie te bestuderen .............................................77
13.4 Genetische manipulatie van zoogdieren met muis als voorbeeld ................................................78
13.4.1 Conventionele/ conditionele KO/ KI.................................................................................... 79
13.4.2 Cre-Lox recombinase systeem .......................................................................................... 80
13.4.3 Celablatie door transgene expressie van difterie toxine receptor ......................................... 82
13.4.4 Gehumaniseerde muizen .................................................................................................. 82
Hoofdstuk 14: Omgevings-biotechnologie in een notendop ........................................................... 83
, 14.1 Witte technologie.....................................................................................................................83
14.2 Blauwe technologie .................................................................................................................84
Hoofdstuk 1: CRISPR/cas nuclease technologie en toepassingen.................................................. 5c
CRISPR cas...................................................................................................................................... 5
CRSPR cas technologie .................................................................................................................... 5
Dubbelstrengbeuk introduceren ....................................................................................................... 6
Cas9 ............................................................................................................................................... 6
Beperkingen transport CRISPR-cas ................................................................................................... 6
Verkrijgen functioneel cas9-sgRNA ribonucleoproteïne in de celkern .................................................. 7
CRISPR cas expressievector (knockout plasmide).............................................................................. 8
BPK764: NHEJ herstel ....................................................................................................................... 9
Transportmechanismen voor aanbreng CRIPR-cas in EU cellen.......................................................... 9
Alternatief gebruik van de CRISPR-cas technologie ...........................................................................10
RNA Editing .....................................................................................................................................11
Aan- en uitschakelen van CRISPR/cas-gene editing ..........................................................................11
Toepassingen CRISPR cas ...............................................................................................................11
Beperkingen van gebruik CRISPR cas ...............................................................................................11
Beperken aspecifieke effecten cas9 .................................................................................................12
Hoofdstuk 2: RNA isolatie ............................................................................................................ 12
2.1 Inleiding RNA – verschillende soorten RNAs ................................................................................12
2.2 RNA isolatiemethoden ...............................................................................................................12
2.2.1 Protocol RNA isolatie .......................................................................................................... 13
Hoofdstuk 3: In vitro cDNA synthese, RACE en miRNA PCR............................................................ 20
3.1 Aanmaak van DNA in vitro ..........................................................................................................20
3.1.1 Chemische DNA synthese ................................................................................................... 20
3.1.2 DNA printing: 3D printer....................................................................................................... 20
3.1.3 Aanmaak cDNA ................................................................................................................... 20
3.1.4 Aanmaak DNA via PCR ........................................................................................................ 22
3.2 Analyse van miRNA door RT-PCR ................................................................................................23
Hoofdstuk 4: Expressie analyse van RNA en DNA .......................................................................... 24
4.1 Kwantitatieve PCR - qPCR ..........................................................................................................24
4.1.1 Algemene detectie strategieën qPCR .................................................................................. 24
4.1.2 Theoretische aspecten qPCPR ............................................................................................ 26
4.1.3 Data-analyse qPCR ............................................................................................................ 27
4.2 Digitale druppel PCR ..................................................................................................................28
, 4.3 nCounter ...................................................................................................................................29
Hoofdstuk 5: Next generation sequentie analyse: DNA en RNA sequeneren ................................... 30
5.1 NGS ..........................................................................................................................................30
5.1.1 Aanmaak bibliotheek & belangrijkste methoden NGS (library prep)........................................ 30
5.1.2 Aanmaak RNA bibliotheek ................................................................................................... 31
5.1.3 Klonale expansie bibliotheek door polony of bridge PCR ....................................................... 33
5.1.4 Sequencing by synthesis ..................................................................................................... 34
5.1.5 NGS data analyse................................................................................................................ 34
5.2 Single cell NG sequencing ..........................................................................................................35
5.3 Spatial transcriptomics ..............................................................................................................36
Hoofdstuk 6: third generation sequencing .................................................................................... 36
6.1 Pacbio Sequel system ................................................................................................................36
6.3 Nanopore syteem ......................................................................................................................38
Hoofdstuk 7: mRNA transcript analyse ......................................................................................... 39
7.1 Inleiding ISH: CISH en FISH ........................................................................................................39
7.2 ViewRNA ISH Cell Assay.............................................................................................................40
7.3 DualRNAscope ISH-IHC .............................................................................................................40
7.4 MerFISH ....................................................................................................................................41
Hoofdstuk 8: RNA technologiën.................................................................................................... 42
8.1 Principe van antisense RNA/ RNA interference ............................................................................42
8.1.1 Aanmaak antisense en RNAi in het lab ................................................................................. 43
8.2 Circulair RNA .............................................................................................................................45
8.4 Long non-coding RNA ................................................................................................................45
Hoofdstuk 9: epigenetisch onderzoek........................................................................................... 46
9.1 Inleiding epigenetica ..................................................................................................................46
9.2 Epigenetische processen ...........................................................................................................47
9.2.1 DNA methylatie ................................................................................................................... 47
9.2.2 Histone modificatie enzymen .............................................................................................. 47
9.3 Technieken om epigenetische veranderingen en histone modificaties te bestuderen ....................47
9.3.1 Methylatiegevoelige restrictie enzymen ................................................................................ 47
9.3.2 Whole genome bisulfiet sequencen ..................................................................................... 48
9.2.3 Mass Array Sequenom epityper ...................................................................................... 49
9.2.4 ChipSeq immunoprecipitatie om histon modificaties te analyseren ................................. 50
9.3 Toepassingen: factoren van invloed op DNA methylatie veranderingen .........................................50
Hoofdstuk 10: Genexpressie in prokaryoten ................................................................................. 52
10.1 Inleiding en basisconcepten .....................................................................................................52
, 10.2 Opbouw prokaryote expressievector .........................................................................................53
10.3 Regulatie expressie met induceerbare bacteriële promotoren ....................................................54
10.4 Problemen en optimalisatie EU eiwitexpressie in PRO cel .........................................................56
10.5 Tags en fusiesystemen .............................................................................................................57
10.6 Courante expressievectoren ....................................................................................................60
10.6.1 pET ................................................................................................................................... 60
10.6.3 pTrcHis vector systeem ..................................................................................................... 61
10.6.3 pBAD/ His fusie-vector met polyHis-tagvectoren ................................................................ 61
10.6.4 pGEX fusie-vectoren met GST ............................................................................................ 61
10.7 Kloneringsmethoden ................................................................................................................62
10.7.1 SLIC ................................................................................................................................. 62
10.7.2 Gateway cloning ............................................................................................................... 62
10.8 Toepassingen ..........................................................................................................................63
Hoofdstuk 11: Genexpressie in Eukaryoten ................................................................................... 64
11.1 Expressie EU in insectencel ......................................................................................................64
11.2 Expressie EU in mammalia of zoogdiercellen .............................................................................64
11.3 Types virale vectoren ...............................................................................................................66
11.3.1 Adenovirus à ................................................................................................................... 66
11.3.2 Adeno-associated virus ..................................................................................................... 68
11.3.3 Retrovirus ......................................................................................................................... 69
11.3.4 Lentivirus .......................................................................................................................... 70
Hoofdstuk 12: functie analyse technieken .................................................................................... 73
12.1 Analyse DNA regulator gebieden ...............................................................................................73
12.1.1 DNAse 1 footprinting ......................................................................................................... 73
12.1.2 Elektroforetische mobiliteit shift assay .............................................................................. 74
12.1.3 ChIP chromatine immunoprecipitatie ................................................................................ 74
12.2 Reporter genen en deletie analyse ............................................................................................75
12.3 Analyse naar de interactie tussen EW .......................................................................................76
Hoofdstuk 13: Genetische modificatie van dieren ......................................................................... 76
13.1 Genetische manipulatie in dieren .............................................................................................76
13.2 Zebravis: model voor onderzoek naar humane ziekten ...............................................................76
13.3 Meest gebruikte methoden om zebravis gen functie te bestuderen .............................................77
13.4 Genetische manipulatie van zoogdieren met muis als voorbeeld ................................................78
13.4.1 Conventionele/ conditionele KO/ KI.................................................................................... 79
13.4.2 Cre-Lox recombinase systeem .......................................................................................... 80
13.4.3 Celablatie door transgene expressie van difterie toxine receptor ......................................... 82
13.4.4 Gehumaniseerde muizen .................................................................................................. 82
Hoofdstuk 14: Omgevings-biotechnologie in een notendop ........................................................... 83
, 14.1 Witte technologie.....................................................................................................................83
14.2 Blauwe technologie .................................................................................................................84
