GENTECHNOLOGIE
3de jaar Biotechnologie 2021 – 2022
1
,INHOUD
Two hybrid technieken _______________________________________________________ 5
Yeast two hybrid ________________________________________________________________ 5
Principe Yeast two hybrid _______________________________________________________________ 5
Werkingsmechanisme Yeast two hybrid ____________________________________________________ 5
Aas – Prooiplasmide _________________________________________________________________ 7
Rapporteergenen ___________________________________________________________________ 8
Auto – activatie _______________________________________________________________________ 8
Bacterial Two Hybrid _____________________________________________________________ 9
Principe Bacterial two hybrid ____________________________________________________________ 9
Voorbeeld Two hybrid toepassingen ___________________________________________________ 10
Display technieken _________________________________________________________ 10
Meest gekende display techniek: Faagdisplay ________________________________________ 10
Faag M13___________________________________________________________________________ 10
Principe van M13 faagdisplay ___________________________________________________________ 12
1) Aanmaak van een faagbank ______________________________________________________ 12
Aanmaak faagbank en faag productie_________________________________________________ 13
2) Affiniteitsselectie_______________________________________________________________ 14
3) Bevestiging van affiniteit: faag ELISA _______________________________________________ 15
Gistdisplay ____________________________________________________________________ 16
Voorbeelden van (faag) display toepassingen ________________________________________ 17
Grootschalige DNA en RNA analyse ____________________________________________ 17
array technologie __________________________________________________________ 17
Soorten microarrys _____________________________________________________________ 18
Gespotte Microarry (Minst gebruikt) _____________________________________________________ 18
In situ gesynthetiseerde arrays __________________________________________________________ 19
Bead arrays _________________________________________________________________________ 19
1-color vs 2-color arrays _____________________________________________________________ 20
Microarry toepassingen _________________________________________________________ 20
Genexpressie profielen (Transcriptomics) _________________________________________________ 20
Genexpressie-analyse: target bereiding _________________________________________________ 21
Comparatieve genomische hybridisatie (Array CGH) _________________________________________ 22
Genotyping arrays (SNP chips) __________________________________________________________ 23
Probe design ________________________________________________________________________ 24
Voorbeelden microarray toepassingen _____________________________________________ 24
High – throughtput sequencing _______________________________________________ 25
Sanger Sequencing _____________________________________________________________ 25
Stappen Sanger sequencing ____________________________________________________________ 25
Next – Generation Sequencing (NGS)_______________________________________________ 26
Verschil tussen Next Generation Sequencing (NGS) en Sanger Sequencing ________________________ 26
Basisprincipe van NGS _________________________________________________________________ 27
2
, Immobilisatie en klonale amplicatie ____________________________________________________ 27
Massively parallel sequencing ___________________________________________________________ 31
Cyclische reversibele terminatie met fluorescente nucleotiden_______________________________ 31
Pyrosequencing ____________________________________________________________________ 32
Halfgeleider sequencing of semiconductor sequencing _____________________________________ 33
Next-generation sequencing ____________________________________________________________ 34
Third – generation sequencing (TGS) _______________________________________________ 34
Single molecule real-time (SMRT) sequencing ______________________________________________ 34
Nanopore sequencing _______________________________________________________________ 35
Voorbeelden van High – throughput sequencing toepassingen __________________________ 36
Plaatsspecifieke genoommanipulatie __________________________________________ 37
Knouk – out en knock – in via homologe recombinatie ________________________________ 37
Knock – out _________________________________________________________________________ 37
Knock – in __________________________________________________________________________ 38
Conditionele knock-out via Cre – Lox recombinatie ___________________________________ 39
Cre – Lox recombinatie ________________________________________________________________ 39
conditionele knock-out – Uitschakelen van een gen _______________________________________ 40
Crispr ________________________________________________________________________ 42
Crispr – Cas9 ________________________________________________________________________ 42
Crispr – Cas9 systeem _______________________________________________________________ 42
Herstelmechanisme van Crispr – Cas9 ____________________________________________________ 43
Crispr Vectoren ______________________________________________________________________ 44
Andere CRISPR systemen ______________________________________________________________ 45
crispr – Cpf1 ______________________________________________________________________ 45
CRISPR – Cas9n (vERhogen specifiteit) __________________________________________________ 46
CRISPR Base editing _________________________________________________________________ 47
Voorbeelden CRISPR – Cas toepassingen ____________________________________________ 47
RNA interference___________________________________________________________ 48
Post – transcriptional gene silencing _______________________________________________ 48
Natuurlijk proces van RNAi _______________________________________________________ 48
RNAi in de biotechnologie _______________________________________________________ 50
Aanmaak van siRNA via in vitro transcriptie ________________________________________________ 50
SIrna vectoren _______________________________________________________________________ 52
Voorbeelden RNAi toepassingen __________________________________________________ 53
Virale transformatie ________________________________________________________ 54
Principe Virale transformatie ___________________________________________________________ 54
Retrovirale vectoren ____________________________________________________________ 54
Retrovirussen (HIV – virus (Lentivirussen)) _________________________________________________ 54
Opbouw genoom retrovirus __________________________________________________________ 55
Productie van retrovirale vectoren _______________________________________________________ 56
Productie retrovirale vectoren via packaging cellijnen ______________________________________ 57
Verloop retrovirale transformatie ________________________________________________________ 58
3
, Adeno – associated viral vectoren (AAV) ____________________________________________ 58
Adeno – associated virus_______________________________________________________________ 58
Adeno – associated virus vectoren _______________________________________________________ 60
Verloop AAV – transformatie _________________________________________________________ 61
Gentherapie op basis van virale vectoren ___________________________________________ 62
Car – T immunotherapie _______________________________________________________________ 63
Genetisch gewijzigde (proef) dieren ___________________________________________ 63
Definities _____________________________________________________________________ 63
Micro-injectie in de bevruchte eicel ________________________________________________ 64
Gebruik van embryonale stamcellen _______________________________________________ 65
Creëren knock – out muis door homologe recombinatie ______________________________________ 65
Kruising voor het creëren van transgene muizen met ES – cellen _______________________________ 66
Conditionele knock – out muizen __________________________________________________ 67
Andere diersoorten _____________________________________________________________ 68
Voorbeelden van genetisch gewijzigde dieren _______________________________________ 68
Begrippenlijst _____________________________________________________________ 69
4
3de jaar Biotechnologie 2021 – 2022
1
,INHOUD
Two hybrid technieken _______________________________________________________ 5
Yeast two hybrid ________________________________________________________________ 5
Principe Yeast two hybrid _______________________________________________________________ 5
Werkingsmechanisme Yeast two hybrid ____________________________________________________ 5
Aas – Prooiplasmide _________________________________________________________________ 7
Rapporteergenen ___________________________________________________________________ 8
Auto – activatie _______________________________________________________________________ 8
Bacterial Two Hybrid _____________________________________________________________ 9
Principe Bacterial two hybrid ____________________________________________________________ 9
Voorbeeld Two hybrid toepassingen ___________________________________________________ 10
Display technieken _________________________________________________________ 10
Meest gekende display techniek: Faagdisplay ________________________________________ 10
Faag M13___________________________________________________________________________ 10
Principe van M13 faagdisplay ___________________________________________________________ 12
1) Aanmaak van een faagbank ______________________________________________________ 12
Aanmaak faagbank en faag productie_________________________________________________ 13
2) Affiniteitsselectie_______________________________________________________________ 14
3) Bevestiging van affiniteit: faag ELISA _______________________________________________ 15
Gistdisplay ____________________________________________________________________ 16
Voorbeelden van (faag) display toepassingen ________________________________________ 17
Grootschalige DNA en RNA analyse ____________________________________________ 17
array technologie __________________________________________________________ 17
Soorten microarrys _____________________________________________________________ 18
Gespotte Microarry (Minst gebruikt) _____________________________________________________ 18
In situ gesynthetiseerde arrays __________________________________________________________ 19
Bead arrays _________________________________________________________________________ 19
1-color vs 2-color arrays _____________________________________________________________ 20
Microarry toepassingen _________________________________________________________ 20
Genexpressie profielen (Transcriptomics) _________________________________________________ 20
Genexpressie-analyse: target bereiding _________________________________________________ 21
Comparatieve genomische hybridisatie (Array CGH) _________________________________________ 22
Genotyping arrays (SNP chips) __________________________________________________________ 23
Probe design ________________________________________________________________________ 24
Voorbeelden microarray toepassingen _____________________________________________ 24
High – throughtput sequencing _______________________________________________ 25
Sanger Sequencing _____________________________________________________________ 25
Stappen Sanger sequencing ____________________________________________________________ 25
Next – Generation Sequencing (NGS)_______________________________________________ 26
Verschil tussen Next Generation Sequencing (NGS) en Sanger Sequencing ________________________ 26
Basisprincipe van NGS _________________________________________________________________ 27
2
, Immobilisatie en klonale amplicatie ____________________________________________________ 27
Massively parallel sequencing ___________________________________________________________ 31
Cyclische reversibele terminatie met fluorescente nucleotiden_______________________________ 31
Pyrosequencing ____________________________________________________________________ 32
Halfgeleider sequencing of semiconductor sequencing _____________________________________ 33
Next-generation sequencing ____________________________________________________________ 34
Third – generation sequencing (TGS) _______________________________________________ 34
Single molecule real-time (SMRT) sequencing ______________________________________________ 34
Nanopore sequencing _______________________________________________________________ 35
Voorbeelden van High – throughput sequencing toepassingen __________________________ 36
Plaatsspecifieke genoommanipulatie __________________________________________ 37
Knouk – out en knock – in via homologe recombinatie ________________________________ 37
Knock – out _________________________________________________________________________ 37
Knock – in __________________________________________________________________________ 38
Conditionele knock-out via Cre – Lox recombinatie ___________________________________ 39
Cre – Lox recombinatie ________________________________________________________________ 39
conditionele knock-out – Uitschakelen van een gen _______________________________________ 40
Crispr ________________________________________________________________________ 42
Crispr – Cas9 ________________________________________________________________________ 42
Crispr – Cas9 systeem _______________________________________________________________ 42
Herstelmechanisme van Crispr – Cas9 ____________________________________________________ 43
Crispr Vectoren ______________________________________________________________________ 44
Andere CRISPR systemen ______________________________________________________________ 45
crispr – Cpf1 ______________________________________________________________________ 45
CRISPR – Cas9n (vERhogen specifiteit) __________________________________________________ 46
CRISPR Base editing _________________________________________________________________ 47
Voorbeelden CRISPR – Cas toepassingen ____________________________________________ 47
RNA interference___________________________________________________________ 48
Post – transcriptional gene silencing _______________________________________________ 48
Natuurlijk proces van RNAi _______________________________________________________ 48
RNAi in de biotechnologie _______________________________________________________ 50
Aanmaak van siRNA via in vitro transcriptie ________________________________________________ 50
SIrna vectoren _______________________________________________________________________ 52
Voorbeelden RNAi toepassingen __________________________________________________ 53
Virale transformatie ________________________________________________________ 54
Principe Virale transformatie ___________________________________________________________ 54
Retrovirale vectoren ____________________________________________________________ 54
Retrovirussen (HIV – virus (Lentivirussen)) _________________________________________________ 54
Opbouw genoom retrovirus __________________________________________________________ 55
Productie van retrovirale vectoren _______________________________________________________ 56
Productie retrovirale vectoren via packaging cellijnen ______________________________________ 57
Verloop retrovirale transformatie ________________________________________________________ 58
3
, Adeno – associated viral vectoren (AAV) ____________________________________________ 58
Adeno – associated virus_______________________________________________________________ 58
Adeno – associated virus vectoren _______________________________________________________ 60
Verloop AAV – transformatie _________________________________________________________ 61
Gentherapie op basis van virale vectoren ___________________________________________ 62
Car – T immunotherapie _______________________________________________________________ 63
Genetisch gewijzigde (proef) dieren ___________________________________________ 63
Definities _____________________________________________________________________ 63
Micro-injectie in de bevruchte eicel ________________________________________________ 64
Gebruik van embryonale stamcellen _______________________________________________ 65
Creëren knock – out muis door homologe recombinatie ______________________________________ 65
Kruising voor het creëren van transgene muizen met ES – cellen _______________________________ 66
Conditionele knock – out muizen __________________________________________________ 67
Andere diersoorten _____________________________________________________________ 68
Voorbeelden van genetisch gewijzigde dieren _______________________________________ 68
Begrippenlijst _____________________________________________________________ 69
4
