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

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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).

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