In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos
Published: April 30, 2023
Abstract
Source: Feng W. et al. CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer. J. Vis. Exp. (2018)
This video demonstrates the delivery of plasmid DNA into cerebellar neuronal cells of murine embryos using in utero electroporation. This method is useful to gain understanding of in vivo function of genes in normal brain development and the changes in gene expression of neuron cells during tumor progression.
Protocol
1. Perform In Utero Electroporation Breed 6 to 8-week-old mice and prepare the tools for in utero electroporation. Cross male homozygous Rosa26-CAG-LSL-Cas9-P2A-EGFP mice with female CD-1 mice. Time the pregnancy of the CD-1 mice from the day the vaginal plug is detected (E0.5). Embryos are electroporated at day E13.5. Pull borosilicate glass capillaries (inside diameter: 0.6–0.8 mm) with a micropipette puller. Use the following setting…
Açıklamalar
The authors have nothing to disclose.
Materials
| Autoclave band | Kisker Biotech | 150262 | |
| Electro Square Porator | BTX | ECM830 | |
| Endofree Maxi Kit | Qiagen | 12362 | |
| Ethanol | Merck | 107017 | |
| Eye ointment (Bepanthen) | Bayer | 81552983 | |
| Fast Green | Merck | 104022 | |
| Filter (0.22 µm) | Merck | F8148 | |
| Forceps straight | Fine Science Tools | 91150-20 | |
| Gauze (X100 ES-pads 8f 10 x 10 cm) | Fisher Scientific | 15387311 | |
| Glass Capillary with Filament | Narishige | GD1-2 | |
| Heating Pad | ThermoLux | 463265 / -67 | |
| Isoflurane | Zoetis | TU061219 | |
| Ring Forceps | Fine Science Tools | 11103-09 | |
| Stereomicroscope | Nikon | C-PS | |
| Surgical scissors | Fine Science Tools | 91460-11 | |
| Surgical scissors with blunt tip | Fine Science Tools | 14072-10 | |
| Tweezers w/5mm- disk electrodes Platinum | Xceltis GmbH | CUY650P5 | |
| Metamizol | WDT | ||
| Non-sterile Silk Suture Thread (0.12 mm) | Fine Science Tools | 18020-50 | |
| PBS (1x) | Life Technologies | 14190169 | |
| Tissue scissors Blunt (11.5 cm) | Fine Science Tools | 14072-10 | |
| pCAG-EGxxFP | Addgene | 50716 | |
| pX330 plasmid | Addgene | 42230 |
Etiketler
Bu Makaleden Alıntı Yapın
In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos. J. Vis. Exp. (Pending Publication), e20685, doi: (2023).