In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos

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

1. 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.
   1. Pull borosilicate glass capillaries (inside diameter: 0.6–0.8 mm) with a micropipette puller. Use the following settings: P = 500, Heat = 560, Pull = 150, Vel = 75, Time = 250. Label the capillary tip with black ink for better visualization during the injection. Trim t....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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