Name: CRISPR-Cas9-gebaseerde mutagenese in de entomopathogene nematode Steinernema hermaphroditum en het onderhoud van mutante lijnen (Video) | JoVE Journal
Uploaded: 2025-12-30T12:55:00.000+00:00
Duration: 7 min 13 s
Description: Leer CRISPR-Cas9-genoombewerking in de entomopathogene nematode Steinernema hermaphroditum. Deze methode maakt het creëren van mutanten mogelijk om genfuncties in symbiose en parasitisme te bestuderen.

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</ol>

<table><tbody><tr><td>Agar (80-100 Mesh)</td><td>Fisher Scientific</td><td>BP26411</td><td>Ook gebruikt in LB Pyruvaat platen</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>16500500</td><td>Ook gebruikt bij genotypering</td></tr><tr><td>Alt-R CRISPR-Cas9 tracrRNA</td><td>IDT</td><td>1072532</td><td></td></tr><tr><td>Alt-R S.p. Cas9 Nuclease V3</td><td>IDT</td><td>1081058</td><td></td></tr><tr><td>Borosilicate naalden</td><td>World Precision Instrument</td><td>1B120F-4</td><td></td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Sigma Aldrich</td><td>C4901</td><td></td></tr><tr><td>Cholesterol</td><td>VWR</td><td>433</td><td>5 mg/mL in ethanol</td></tr><tr><td>Comamonas aquatica (DA1877)</td><td></td><td></td><td></td></tr><tr><td>Dekglas voor agarose pad</td><td>Brain research laboratories</td><td>#4860-1D</td><td>48 x 60 mm no1 dikte</td></tr><tr><td>CRISPRScan</td><td></td><td></td><td>https://www.crisprscan.org/</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>472301</td><td>0.5 M</td></tr><tr><td>DNA Clean and Concentrator-5</td><td>Zymo Research</td><td>D4014</td><td></td></tr><tr><td>FemtoJet 4x Microinjector met Grip head set 4 maat 0 naaldhouder</td><td>Eppendorf/Calibre</td><td>5253000017</td><td></td></tr><tr><td>Galleria melonella 5e instar larven</td><td>GrubCo</td><td></td><td>https://www.grubco.com/index.cfm</td></tr><tr><td>Gel beeldsysteem</td><td>Azure400</td><td>AZI400-01</td><td></td></tr><tr><td>Gel laaddye</td><td>Thermo Fisher Scientific</td><td>B72</td><td></td></tr><tr><td>Gelatine (2%)</td><td>Sigma Aldrich</td><td>G1393</td><td></td></tr><tr><td>GelRed</td><td>Biotium</td><td>#41003</td><td></td></tr><tr><td>Genotyperingsprimers: DNA oligo, 25 nmol</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Gibco Bacto Peptone</td><td>Fisher Scientific</td><td>DF0118-17-0</td><td></td></tr><tr><td>Gibco Bacto Tryptone</td><td>Fisher Scientific</td><td>DF0123-17-3</td><td>Ook gebruikt in Dark LB, LB Pyruvaat platen</td></tr><tr><td>Gibco Bacto Yeast Extract</td><td>Fisher Scientific</td><td>DF0127-17-9</td><td>Ook gebruikt in Dark LB, LB Pyruvaat platen</td></tr><tr><td>Grip head set 4 maat 1 naaldhouder</td><td>Calibre</td><td>EPE-5196083008</td><td>(gepaard met borosilicaatnaald 1B120F-4)</td></tr><tr><td>Halocarbon olie 700</td><td>Sigma Aldrich</td><td>H8898</td><td></td></tr><tr><td>Harde platina draaddraad voor dagelijks onderhoud</td><td>Tritech Research</td><td>PT-9901</td><td></td></tr><tr><td>Inference of CRISPR edits (ICE)</td><td>Synthego</td><td>N/A</td><td>Software gebruikt om Sangersequenties te analyseren om indels te identificeren</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Laser naald puller P-2000</td><td>Sutter</td><td>P-2000G</td><td></td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Sigma Aldrich</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate (MgSO4)</td><td>Sigma Aldrich</td><td>M7506</td><td>Voor M9 buffer, Ook gebruikt in NGM</td></tr><tr><td>Microloader tip 2x96ST</td><td>Calibre</td><td>930001007</td><td></td></tr><tr><td>Micromanipulator Montage Adapter voor Zeiss Axio Observer en Axiovert 200</td><td>Tritech Research</td><td>NZ-19-2</td><td></td></tr><tr><td>Microscoop</td><td>Tritech</td><td>SMT1</td><td></td></tr><tr><td>Nicotine</td><td>Sigma Aldrich</td><td>N3876</td><td></td></tr><tr><td>NP-40</td><td>Thermo Fisher Scientific</td><td>85124</td><td></td></tr><tr><td>Nuclease Free Duplex Buffer</td><td>IDT</td><td>11-01-03-01</td><td></td></tr><tr><td>OneTaq MasterMix</td><td>NEB</td><td>M0486</td><td></td></tr><tr><td>Pipet opslagdoos</td><td>Sutter Instruments</td><td>BX10</td><td></td></tr><tr><td>Kaliumchloride (KCl)</td><td>Sigma Aldrich</td><td>793590</td><td></td></tr><tr><td>Kaliumfosfaat, dibasisch (K2HPO4)</td><td>Sigma Aldrich</td><td>P3786</td><td>KOH voor NGM</td></tr><tr><td>Kaliumfosfaat, monobasisch (KH2PO4)</td><td>Sigma Aldrich</td><td>P5655</td><td>Voor M9 buffer, Ook gebruikt in NGM</td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>P6556</td><td></td></tr><tr><td>Quarts naalden</td><td>Sutter Instruments</td><td>QF100-70-10</td><td></td></tr><tr><td>S. hermaphroditium genoom</td><td>NCBI</td><td></td><td>https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_030435675.1/</td></tr><tr><td>sh-unc-22 Alt-R CRISPR-Cas9 crRNA's</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Natriumchloride (NaCl)</td><td>VWR</td><td>SS0430</td><td>Ook gebruikt in M9, Dark LB, LB Pyruvaat platen</td></tr><tr><td>Natriumfosfaat, dibasisch (Na2HPO4.7H20)</td><td>Sigma Aldrich</td><td>S9390</td><td>Voor M9 buffer</td></tr><tr><td>Natriumpyruvaat</td><td>Sigma Aldrich</td><td>P2256</td><td>Ook gebruikt in LB Pyruvaat platen</td></tr><tr><td>Zachte platina draaddraad voor micro-injectie</td><td>Surepure Chemetals</td><td>6824</td><td>Platinum 90%, Iridium 10% legeringdraad, 0,002 inch diameter x 5 ft lang</td></tr><tr><td>Standaard naaldhouder</td><td>Tritech Research</td><td>HI-7</td><td></td></tr><tr><td>Steinernema hermaphroditum</td><td></td><td></td><td></td></tr><tr><td>Stereo microscoop</td><td>Leica</td><td>Inveta 3</td><td></td></tr><tr><td>Trehalose</td><td>Cole-Parmer</td><td>EW-88195-99</td><td>0,08 M</td></tr><tr><td>Tris</td><td>Fisher Scientific</td><td>AM9855G</td><td>pH 8,0</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>Verticale Micromanipulator Montage Adapter</td><td>Tritech Research</td><td>NR2</td><td></td></tr><tr><td>Xenorhabdus griffiniae (HGB2511)</td><td></td><td></td><td></td></tr></tbody></table>

crispr-cas9-gebaseerde mutagenese in de entomopathogene nematode steinernema hermaphroditum en het onderhoud van mutante lijnen

CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines

CRISPR-Cas9-gebaseerde mutagenese in de entomopathogene nematode Steinernema hermaphroditum en het onderhoud van mutante lijnen

Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus ...

crispr-cas9-based-mutagenesis-entomopathogenic-nematode-steinernema

Research

JoVE Journal

Genetics

Genetica

Screening voor mutanten met fenotypische afwijkingen is een eenvoudige methode voor het identificeren van genen die functie in een bepaald biologisch proces. In dit artikel beschrijven we hoe u de cultuur vrij levende wormen (bv.

Een inleiding tot Worm Lab: van kweken van Worms naar Mutagenesis

Hier presenteren we een methode om het genoom van C. elegans ingenieur met behulp van CRISPR-Cas9 ribonucleoproteins en homologie afhankelijke reparatie sjablonen.

Een snelle en Facile pijpleiding voor het genereren van Genomic punt mutanten in C. elegans met behulp van CRISPR/Cas9 Ribonucleoproteins

Hier beschrijven we een protocol om een gen knock-out in de extracellulaire amastigote van Trypanosoma cruzi te introduceren, met behulp van het crispr/Cas9-systeem. De groei fenotype kan worden opgevolgd door de celtelling van de Axenische amastigote cultuur of door proliferatie van intracellulaire amastigotes na de invasie van de cel van de host.

Introductie van een gen knock-out direct in het Amastigote stadium van Trypanosoma cruzi met behulp van het crispr/Cas9 systeem

Dit protocol beschrijft micro-injectieprocedures voor Culex quinquefasciatus embryo's die zijn geoptimaliseerd om te werken met CRISPR/Cas9 genbewerkingstools. Deze techniek kan efficiënt site-specifieke, erfelijke, kiembaanmutaties genereren die kunnen worden gebruikt voor het bouwen van genetische technologieën in deze onderbelichte ziektevector.

Embryo micro-injection technieken voor efficiënte site-specifieke mutagenese in Culex quinquefasciatus

CRISPR/Cas9 wordt steeds vaker gebruikt om genfunctie in niet-modelorganismen te karakteriseren. Dit protocol beschrijft hoe knock-out lijnen van Culex pipienste genereren, van het bereiden van injectiemengsels tot het verkrijgen en injecteren van muggenembryo's, evenals hoe geïnjecteerde muggen en hun nakomelingen op te wekken, te kruisen en te screenen op gewenste mutaties.

Embryo's van Culex-muggen voorbereiden en injecteren om null-mutaties te genereren met CRISPR/Cas9

Dit protocol beschrijft de stappen van CRISPR/Cas9 gerichte mutagenese bij zandvliegen: embryoverzameling, injectie, insectenopfok en identificatie, evenals selectie van mutaties van belang.

Zandvlieg (Phlebotomus papatasi) Embryo Microinjection voor CRISPR/Cas9 Mutagenese

Veel kenmerken van insectenesocialiteit zijn afhankelijk van communicatie binnen de kolonie en arbeidsverdeling. Genetische manipulatie van belangrijke regulerende genen in mierenembryo's via micro-injectie en CRISPR-gemedieerde mutagenese geeft inzicht in de aard van altruïstisch gedrag bij eusociale insecten.

Embryo-injecties voor CRISPR-gemedieerde mutagenese in de mier Harpegnathos saltator

Het doel van dit protocol is om naadloze CRISPR/Cas9-bewerking van het C. elegans-genoom mogelijk te maken met behulp van geassembleerde ribonucleoproteïnecomplexen en de dpy-10 co-CRISPR-marker voor screening. Dit protocol kan worden gebruikt om een verscheidenheid aan genetische modificaties in C. elegans te maken, waaronder inserties, deleties, genvervangingen en codonsubstituties.

CRISPR/Cas9 Editing van het C. elegans rbm-3.2 Gen met behulp van de dpy-10 Co-CRISPR Screening Marker en Geassembleerde Ribonucleoproteïne Complexen.

Hier wordt een protocol gepresenteerd van Helicoverpa armigera (Hübner) embryo micro-injectie en knock-out mutant identificatie gemaakt door CRISPR / Cas9 genoombewerking. Gemuteerde insecten maken verder onderzoek naar genfunctie en interactie tussen verschillende genen in vivo mogelijk.

Embryo Microinjectie en Knockout Mutant Identificatie van CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Deze studie biedt een systematisch geoptimaliseerde procedure van CRISPR/Cas9 ribonuclease-gebaseerde constructie van homozygote sprinkhanenmutanten en een gedetailleerde methode voor cryopreservatie en reanimatie van de sprinkhaneneieren.

Constructie van homozygote mutanten van trekkende sprinkhanen met behulp van CRISPR/Cas9-technologie

Entomopathogene nematoden leven in symbiose met bacteriën en samen infecteren ze met succes insecten door hun aangeboren immuunsysteem te ondermijnen. Om onderzoek naar de genetische basis van nematodeninfectie te bevorderen, worden methoden beschreven voor het handhaven en genetisch manipuleren van entomopathogene nematoden.

Kweken en genetisch manipuleren van entomopathogene nematoden

Leer CRISPR-Cas9-genoombewerking in de entomopathogene nematode Steinernema hermaphroditum. Deze methode maakt het creëren van mutanten mogelijk om genfuncties in symbiose en parasitisme te bestuderen.

CRISPR-Cas9-gebaseerde mutagenese in de entomopathogene nematode Steinernema hermaphroditum en het onderhoud van mutante lijnen

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