Gonad Microinjection: En metode til sammensat levering direkte ind i kimen af C. elegans

Abstract

Kilde: Farboud, B. et al, Enhanced Genome Redigering med Cas9 Ribonucleoprotein i forskellige celler og organismer. J. Vis. Exp. (2018). 

Denne video beskriver microinjection, en fælles metode til at skabe transgene C. elegans stammer.  I eksemplet vil vi se microinjection bruges til at introducere et ribonukleoprotein (RNP) kompleks til CRISPR-baser genredigering.

Protocol

Følgende protokol er et uddrag fra Farboud et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms, J. Vis. Exp. (2018). 1. RNP Forsamling Design eksperimentet i god tid, og anskaffe alle RNA-, DNA- og proteinkomponenter på forhånd. Som et første gennemløb kan du prøve en af de positive kon…

Representative Results

Tabel 1: Positiv kontrol med foreløbige genomredigeringseksperimenter. Denne tabel viser de vigtigste oplysninger, der er nødvendige for at udføre et første gangs genomredigeringseksperiment i hver af de celler og organismer, der er beskrevet i denne protokol. Efter disse parametre vil sandsynligvis give et vellykket resultat, der kan bruges til at teste protokollen eller som en baseline til sammenlign…

Materials

Reagents/Materials
DNA oligonucleotides	Integrated DNA Technologies	–	IDT will provide custom DNA sequences, including those in Table 1
Guide RNAs	Synthego	–	Synthego will provide high-quality sgRNAs for S. pyogenes Cas9, including custom sgRNAs containing the targeting sequences included in Table 1
Purified Cas9 protein (EnGen Cas9 NLS, S. pyogenes)	New England Biosciences	M0646T	If possible, purifying Cas9 in-house or purchasing from local core facilities is a less expensive option
OP50 Escherichia coli	Caenorhabditis Genetics Center	OP-50	https://cgc.umn.edu/
Nematode Growth Media agar in petri dishes	–	–	See Stiernagle, T (ref. 59)
Standard borosilicate glass capillaries with filament:  4 in (100 mm), 1/0.58 OD/ID	World Precision Instruments	1B100F-4
Single-barrel standard borosilicate glass capillaries: 6 in (152 mm), 2/1.12 OD/ID	World Precision Instruments	1B200-6
Cover glass; 24 × 50 mm	Thermo Fisher Scientific	12-544E
Cover glass; 22 × 22 mm	Thermo Fisher Scientific	12-518-105K
Apex LE agarose	Genesee Scientific	20-102
Halocarbon oil 700	Sigma-Aldrich	H8898-100ML
pCFJ90 plasmid	Addgene	19327
Capillary tubes with filament: 4 in (1.0 mm)	World Precision Instruments	T2100F-4
Petri dishes (100 × 15 mm)	–
9" pasteur pipettes	–
Nuclease-free water	–
Equipment
MZ75 Stereomicroscope	Leica	Out-of-production. Current model is the M80 Stereomicroscope
Axio Vert35 inverted phase contrast fluorescent microscope	Zeiss	Out-of-production. Current model is the Axio VertA.1
Laser-based micropipette puller (for C. elegans protocol)	Sutter Instrument	FG-P2000
Picoliter Microinjector (for C. elegans protocol)	Warner Instruments	PLI-100A
Three-axis Joystick oil hydraulic micromanipulator	Narishige International	MO-202U
Coarse manipulator	Narishige International	MMN-1
Microloader pipette tips	Eppendorf	5242956003
NG-agar