In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Summary

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Abstract

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Introduction

The neocortex is the outer sheet of the mammalian cerebrum and the seat of higher cognitive functions^1,2,3,4,5. In order to achieve an acute genetic manipulation in the mammalian neocortex in vivo during the embryonic development, two different methods have been explored: viral infection⁶ and in utero electroporation⁷. Both methods allow efficient targeting of neocortical cells but suffer from some limitations. The major advantage of in utero electroporation c....

Protocol

All experimental procedures were conducted in agreement with the German Animal Welfare Legislation after approval by the Landesdirektion Sachsen (licenses TVV 2/2015 and TVV 21/2017).

1. Preparation for in utero electroporation

1. Prepare the DNA mixture. In this protocol a final concentration of 1 µg/µL of pGFP is used. Dissolve DNA in PBS and supplement with 0.1% Fast Green to facilitate visualization. Once prepared, mix the DNA mixture by pipetting up and down several tim.......

Discussion

In utero electroporation in ferret is an important technique, with advantages and disadvantages with respect to other methods. There are critical steps and limitations to this method, as well as potential modifications and future applications to keep in mind.

Since the pioneering work of Victor Borrell and colleagues on genetic manipulation of the postnatal ferret neocortex via electroporation or viral injection^35,42,

Materials

Name	Company	Catalog Number	Comments
1ml syringe	BD	309628	Electroporation
4-0 Vicryl suture	Ethicon	V392ZG	Surgery
Aluminium spray	cp-pharma	98017	Surgery
Amoxicilin+clavulanic acid (Synulox RTU)	WDT	6301	Surgery
Cappilary holder	WPI	MPH6S12	Electroporation
Dexpanthenol Ointment solution	Bayer	6029009.00.00	Surgery
Drape sheet 45x75cm	Hartmann	2513052	Surgery
Electrode Tweezer, platinum plated 5mm	BTX	45-0489	Electroporation
Electroporator	BTX	ECM830	Electroporation
Fast Green	Sigma	F7258-25G	Electroporation
Ferret Mustela putorius furo	Marshall	NA	Experimental organism
Fiber optic light source	Olympus	KL1500LCD	Electroporation
Forceps	Allgaier instrumente	08-033-130	Surgery
Forceps 3C-SA	Rubis Tech	3C-SA	Surgery
Forceps 55	Dumostar	11295-51	Surgery
Forceps 5-SA	Rubis Tech	5-SA	Surgery
Gauze swabs large	Hartmann	401723	Surgery
Gauze swabs small	Hartmann	401721	Surgery
GFAP antibody	Dako	Z0334	Antibody
GFP antibody	Aves labs	GFP1020	Antibody
Glass cappilaries (Borosilicate glass with filament, OD:1.2mm, ID: 0.69mm, 10cm length)	Sutter Instrument	BF120-69-10	Electroporation
Glucose	Bela-pharm	K4011-02	Surgery
Heat pad	Hans Dinslage	Sanitas SHK18	Surgery
Iodine (Betadine solution 100 mg/ml)	Meda	997437	Surgery
Isofluran	CP	21311	Surgery
Loading tips 20µl	Eppendorf	#5242 956.003	Electroporation
Metamizol	WDT	99012	Surgery
Metzenbaum dissecting scissors	Aesculap	BC600R	Surgery
Micropipette puller	Sutter Instrument	Model P-97	Electroporation
pCAGGS-GFP	NA	NA	From Kalebic et al., eLife, 2018
PCNA antibody	Millipore	CBL407	Antibody
pH3 antibody	Abcam	ab10543	Antibody
Scalpel	Aesculap	294200104	Surgery
Shaver	Braun	EP100	Surgery
Sox2 antibody	R+D Systems	AF2018	Antibody
Surgical clamp 13cm	WDT	27080	Surgery
Surgical double spoon (Williger)	WDT	27232	Surgery
Surgical drape	WDT	28800	Surgery
Surgical scissors small	FST	14090-09	Surgery
Suturing needle holder	Aesculap	BM149R	Surgery
Tbr2 antibody	Abcam	ab23345	Antibody
Transfer pipette 3ml	Fischer scientific	13439108	Surgery
Water bath	Julabo	TW2	Surgery