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Samenvatting Gentechnologie

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Een eerste deel omvat een omschrijving van het kloneren van genomisch DNA. De verschillende vectoren die hiervoor gebruikt kunnen worden, worden besproken. In een volgend hoofdstuk wordt een elegante techniek aangeleerd die in staat is eiwitinteragerende partners op te sporen : de ” two hybrid”...

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  • January 22, 2022
  • 70
  • 2021/2022
  • Summary
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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




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