GENTECHNOLOGIE
Theorie
2024-2025
FARMACEUTISCHE EN BIOLOGISCHE LABORATORIUM TECHNOLOGIE
Lowie Boels
,Lowie Boels Gentechnologie: theorie
1. CRISPR/Cas nuclease system ...........................................................................................8
Inleiding ..............................................................................................................................8
Mechanisme bij genetische modificatie in biotechnologie .....................................................8
Stap I: dubbelstrengige breuk (DSB) induceren ..................................................................8
Stap II: DNA herstelmechanisme ......................................................................................9
Het transport van CRISPR/Cas complex tot specifieke doelcellen ..........................................9
Strategie I: DNA-plasmide.................................................................................................9
Strategie II: mRNA en sgRNA cargo .................................................................................. 10
Strategie III: Cas9-sgRNA ribunucleoproteïne .................................................................. 10
Niet virale transportmechanisme .................................................................................... 11
Micro-injectie ............................................................................................................. 11
Elektroporatie ............................................................................................................. 11
Mechanische celdeformatie ........................................................................................ 11
Hydrodynamische injectie ........................................................................................... 11
Nanopartikels ............................................................................................................. 11
Alternatief gebruik van CRISPR/Cas technologie ................................................................. 11
Gen expressie moduleren ............................................................................................... 11
Repressie ................................................................................................................... 11
Activatie ..................................................................................................................... 11
DCas9 ........................................................................................................................ 11
Epigenomische veranderingen..................................................................................... 11
Visualiseren van levende cellen ................................................................................... 12
RNA targetting ................................................................................................................... 12
Gebruik van PAMmer om RNA te editeren ........................................................................ 12
Aan en uitschakelen van CRISPR/Cas gene editing .............................................................. 12
Toepassingen van CRISPR/Cas technologie ........................................................................ 12
Onderzoek ..................................................................................................................... 12
Kliniek ............................................................................................................................ 12
Gene drive...................................................................................................................... 13
Genoom editering in zebravis .......................................................................................... 13
Genoom editering in varkens........................................................................................... 13
Xenotransplantatie ......................................................................................................... 13
Genezen van patiënten ................................................................................................... 13
Mogelijke beperkingen van CRISPR/Cas .............................................................................. 14
2. RNA isolatie ................................................................................................................... 15
Inleiding RNA ..................................................................................................................... 15
1
,Lowie Boels Gentechnologie: theorie
RNA isolatietechnieken ...................................................................................................... 15
Stap I: voorbereiding en staalcollectie ............................................................................. 15
Stap II: Homogenisatie ................................................................................................... 16
Keuze lysisbuffer......................................................................................................... 16
Keuze lysismethode .................................................................................................... 16
Optionele stappen tijdens homogenisatie .................................................................... 16
Stap III A: organische extractie methode voor totaal RNA isolatie...................................... 17
Stap IIIB: silica membraan gebaseerde spin kolom methode voor RNA isolatie ................. 18
Stap IIIC: gebruik van paramagnetische parels voor RNA isolatie ...................................... 18
Stap IV: kwaliteit en kwantiteit controle ........................................................................... 19
Kwantiteit bepalen ...................................................................................................... 19
Kwantiteit en RNA integriteit bepalen ........................................................................... 19
Stap V: Stockeren van RNA ............................................................................................. 20
3. In vitro (c)DNA synthese en RACE .................................................................................... 21
Aanmaak van DNA in vitro .................................................................................................. 21
Chemische DNA synthese .............................................................................................. 21
DNA printen: 3D printing technology ............................................................................... 21
Aanmaak van cDNA ........................................................................................................ 21
Proces van cDNA synthese .......................................................................................... 22
3 enzymen betrokken bij cDNA synthese ...................................................................... 22
Startmolecule om cDNA te synthetiseren .................................................................... 22
Aanmaak van cDNA banken ........................................................................................ 23
Aanmaak van DNA via PCR ............................................................................................. 23
Opsporen van ontbrekende sequentie mbv RAcE ................................................................ 24
Analyse van miRNA door RT-PCR ........................................................................................ 25
4. Expressie analyse van RNA en DNA .................................................................................... 26
Kwantitatieve PCR = qPCR .................................................................................................. 26
Wat is real time PCR? ..................................................................................................... 26
Startmateriaal qPCR ....................................................................................................... 26
Detectiestrategieën ........................................................................................................ 26
SYBR Green/EvaGreen ................................................................................................ 26
TaqMan probe ............................................................................................................. 27
Molecular beacons ..................................................................................................... 27
Dark Quencher ........................................................................................................... 28
Programma van de qPCR ................................................................................................ 28
Theoretische aspecten van qPCR .................................................................................... 29
2
, Lowie Boels Gentechnologie: theorie
Negatieve controles........................................................................................................ 29
Data analyse van qPCR ................................................................................................... 29
MIQE richtlijnen .......................................................................................................... 29
Genexpressie analyse ................................................................................................. 31
Digitale druppel ................................................................................................................. 33
Principe ......................................................................................................................... 33
nCounter technologie ........................................................................................................ 34
5. Next generation sequencing van gDNA & transcripten ..................................................... 35
Next gen sequencing (NGS) ................................................................................................ 35
STAP 1. Aanmaak bibliotheek & belangrijke methode NGS ............................................... 35
1A. Aanmaak van DNA bibliotheek............................................................................... 35
1B. Aanmaak van een RNA bibliotheek......................................................................... 37
STAP 2. Klonale expansie NGS technieken ....................................................................... 39
STAP 3. Sequencing by synthesis = parallel sequeneren ................................................... 41
STAP 4. NGS data analyse en bio-informatica .................................................................. 42
Single cell NGS .................................................................................................................. 43
10x genomics platform met library prep........................................................................... 43
CITE sequenering ........................................................................................................... 44
Spatiale transcriptomics .................................................................................................... 45
Laser capture microdisscetion sequencing ..................................................................... 45
In situ capture sequenering............................................................................................. 46
6. Third generation sequencing ........................................................................................... 47
PacBio sequel system ........................................................................................................ 47
Nanopore technologies ...................................................................................................... 49
7. mRNA transcript analyse .................................................................................................... 51
Inleiding ............................................................................................................................ 51
View RNA ISH cell assay ..................................................................................................... 51
Dual RNAscope ISH-IHC .................................................................................................... 52
Multiplexed error robust FISH = MerFISH ............................................................................. 53
8. RNA technologieën ........................................................................................................... 54
Piwi interagerend RNA ~ PiRNA ........................................................................................... 54
Transposon .................................................................................................................... 54
Aanmaak van piwi interagerende RNA’s ........................................................................... 54
Transcriptionele repressie door piwi pi RNA complex ....................................................... 54
Toepassingen ................................................................................................................. 54
Antisense RNA ................................................................................................................... 54
3
Theorie
2024-2025
FARMACEUTISCHE EN BIOLOGISCHE LABORATORIUM TECHNOLOGIE
Lowie Boels
,Lowie Boels Gentechnologie: theorie
1. CRISPR/Cas nuclease system ...........................................................................................8
Inleiding ..............................................................................................................................8
Mechanisme bij genetische modificatie in biotechnologie .....................................................8
Stap I: dubbelstrengige breuk (DSB) induceren ..................................................................8
Stap II: DNA herstelmechanisme ......................................................................................9
Het transport van CRISPR/Cas complex tot specifieke doelcellen ..........................................9
Strategie I: DNA-plasmide.................................................................................................9
Strategie II: mRNA en sgRNA cargo .................................................................................. 10
Strategie III: Cas9-sgRNA ribunucleoproteïne .................................................................. 10
Niet virale transportmechanisme .................................................................................... 11
Micro-injectie ............................................................................................................. 11
Elektroporatie ............................................................................................................. 11
Mechanische celdeformatie ........................................................................................ 11
Hydrodynamische injectie ........................................................................................... 11
Nanopartikels ............................................................................................................. 11
Alternatief gebruik van CRISPR/Cas technologie ................................................................. 11
Gen expressie moduleren ............................................................................................... 11
Repressie ................................................................................................................... 11
Activatie ..................................................................................................................... 11
DCas9 ........................................................................................................................ 11
Epigenomische veranderingen..................................................................................... 11
Visualiseren van levende cellen ................................................................................... 12
RNA targetting ................................................................................................................... 12
Gebruik van PAMmer om RNA te editeren ........................................................................ 12
Aan en uitschakelen van CRISPR/Cas gene editing .............................................................. 12
Toepassingen van CRISPR/Cas technologie ........................................................................ 12
Onderzoek ..................................................................................................................... 12
Kliniek ............................................................................................................................ 12
Gene drive...................................................................................................................... 13
Genoom editering in zebravis .......................................................................................... 13
Genoom editering in varkens........................................................................................... 13
Xenotransplantatie ......................................................................................................... 13
Genezen van patiënten ................................................................................................... 13
Mogelijke beperkingen van CRISPR/Cas .............................................................................. 14
2. RNA isolatie ................................................................................................................... 15
Inleiding RNA ..................................................................................................................... 15
1
,Lowie Boels Gentechnologie: theorie
RNA isolatietechnieken ...................................................................................................... 15
Stap I: voorbereiding en staalcollectie ............................................................................. 15
Stap II: Homogenisatie ................................................................................................... 16
Keuze lysisbuffer......................................................................................................... 16
Keuze lysismethode .................................................................................................... 16
Optionele stappen tijdens homogenisatie .................................................................... 16
Stap III A: organische extractie methode voor totaal RNA isolatie...................................... 17
Stap IIIB: silica membraan gebaseerde spin kolom methode voor RNA isolatie ................. 18
Stap IIIC: gebruik van paramagnetische parels voor RNA isolatie ...................................... 18
Stap IV: kwaliteit en kwantiteit controle ........................................................................... 19
Kwantiteit bepalen ...................................................................................................... 19
Kwantiteit en RNA integriteit bepalen ........................................................................... 19
Stap V: Stockeren van RNA ............................................................................................. 20
3. In vitro (c)DNA synthese en RACE .................................................................................... 21
Aanmaak van DNA in vitro .................................................................................................. 21
Chemische DNA synthese .............................................................................................. 21
DNA printen: 3D printing technology ............................................................................... 21
Aanmaak van cDNA ........................................................................................................ 21
Proces van cDNA synthese .......................................................................................... 22
3 enzymen betrokken bij cDNA synthese ...................................................................... 22
Startmolecule om cDNA te synthetiseren .................................................................... 22
Aanmaak van cDNA banken ........................................................................................ 23
Aanmaak van DNA via PCR ............................................................................................. 23
Opsporen van ontbrekende sequentie mbv RAcE ................................................................ 24
Analyse van miRNA door RT-PCR ........................................................................................ 25
4. Expressie analyse van RNA en DNA .................................................................................... 26
Kwantitatieve PCR = qPCR .................................................................................................. 26
Wat is real time PCR? ..................................................................................................... 26
Startmateriaal qPCR ....................................................................................................... 26
Detectiestrategieën ........................................................................................................ 26
SYBR Green/EvaGreen ................................................................................................ 26
TaqMan probe ............................................................................................................. 27
Molecular beacons ..................................................................................................... 27
Dark Quencher ........................................................................................................... 28
Programma van de qPCR ................................................................................................ 28
Theoretische aspecten van qPCR .................................................................................... 29
2
, Lowie Boels Gentechnologie: theorie
Negatieve controles........................................................................................................ 29
Data analyse van qPCR ................................................................................................... 29
MIQE richtlijnen .......................................................................................................... 29
Genexpressie analyse ................................................................................................. 31
Digitale druppel ................................................................................................................. 33
Principe ......................................................................................................................... 33
nCounter technologie ........................................................................................................ 34
5. Next generation sequencing van gDNA & transcripten ..................................................... 35
Next gen sequencing (NGS) ................................................................................................ 35
STAP 1. Aanmaak bibliotheek & belangrijke methode NGS ............................................... 35
1A. Aanmaak van DNA bibliotheek............................................................................... 35
1B. Aanmaak van een RNA bibliotheek......................................................................... 37
STAP 2. Klonale expansie NGS technieken ....................................................................... 39
STAP 3. Sequencing by synthesis = parallel sequeneren ................................................... 41
STAP 4. NGS data analyse en bio-informatica .................................................................. 42
Single cell NGS .................................................................................................................. 43
10x genomics platform met library prep........................................................................... 43
CITE sequenering ........................................................................................................... 44
Spatiale transcriptomics .................................................................................................... 45
Laser capture microdisscetion sequencing ..................................................................... 45
In situ capture sequenering............................................................................................. 46
6. Third generation sequencing ........................................................................................... 47
PacBio sequel system ........................................................................................................ 47
Nanopore technologies ...................................................................................................... 49
7. mRNA transcript analyse .................................................................................................... 51
Inleiding ............................................................................................................................ 51
View RNA ISH cell assay ..................................................................................................... 51
Dual RNAscope ISH-IHC .................................................................................................... 52
Multiplexed error robust FISH = MerFISH ............................................................................. 53
8. RNA technologieën ........................................................................................................... 54
Piwi interagerend RNA ~ PiRNA ........................................................................................... 54
Transposon .................................................................................................................... 54
Aanmaak van piwi interagerende RNA’s ........................................................................... 54
Transcriptionele repressie door piwi pi RNA complex ....................................................... 54
Toepassingen ................................................................................................................. 54
Antisense RNA ................................................................................................................... 54
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