Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans
Published: April 30, 2023
Abstract
Source: Farboud, B. et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms. J. Vis. Exp. (2018).
This video describes microinjection, a common method of creating transgenic C. elegans strains. In the example, we will see microinjection used to introduce a ribonucleoprotein (RNP) complex for CRISPR-bases gene editing.
Protocol
The following protocol is an excerpt from Farboud et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms, J. Vis. Exp. (2018). 1. RNP Assembly Design the experiment well in advance, acquiring all the RNA, DNA, and protein components ahead of time. As a first pass, try one of…
Representative Results
Table 1: Positive controls for preliminary genome editing experiments. This table shows the key information needed to perform a first-time genome editing experiment in each of the cells and organisms described in this protocol. Following these parameters is likely to yield a successful result that can be used to test the protocol or as a baseline for comparison once the experimenter is targeting a…
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 |
Cite This Article
Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans. J. Vis. Exp. (Pending Publication), e20168, doi: (2023).