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NEPA21 / CUY21 Applications

[Cultured Embryo] Electroporation for mammalian embryos in the whole embryo culture system

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Procedure

1 Pre-culture embryos in the whole embryo culture system for 1.5-2 hours prior to electroporation.

2 Place the embryo in a Petri dish with Tyrode’s solution

3 Inject 0.1-0.5 µl of plasmid DNA into the brain ventricle with a fine capillary.

4 Apply square pulses using the electroporator (CUY21) and electrodes (chamber-type and forceps-type electrodes are available). 70V, 50 msec at 1-second interval, five pulses are applied for E10.5 mouse embryos.

5 Culture the electroporated embryos for 24-48 hours in the whole embryo culture system.

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Transfection of a fluorescent protein-expression vector into the developing rat cortex.

(A) EGFP-expression vector was electroporated into E11.5 rat telencephalon. The electroporated embryo was cultured in the whole embryo culture system (WEC).

(B) 24 hours after electroporation, EGFP-expression was specifically detected at the dorsal part of the telencephalon.

Tel: telencephalon;

Di: diencephalon

Transfection of fluorescent protein-expression vectors into the rat spinal cord.

(A) EGFP-expression vector was transfected into the spinal cord of E12.5 rat embryos by electroporation. The electroporated embryos were cultured for 24 hours in the whole embryo culture system (WEC).

(B-E) Time-lapse analysis of neuroepithelial cells in the slice culture system.  Histon-EGFP- and DsRed2-expression vectors were co-electroporated into the E12.0 rat spinal cord. The electroporated embryo was cultured for 24 hours in the WEC, then the spinal cord was sliced and time-lapse recording was performed.  (B-E) Cell nuclei (B, green) and cytoplasms (C, magenta) of neuroepithelial cells in the slice are simultaneously labeled by the electroporation (E).

(D) A DIC image.

hb: hindbrain; sc: spinal cord; fl: forelimb; DIC: differential interference contrast

Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine

Masanori Takahashi, Tadashi Nomura, Noriko Osumi *Differentiation, Volume 70, Issue 4-5, Pages 155-162, June 2002