Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Summary

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Abstract

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Introduction

Genetic manipulation of the embryonic mouse brain is a preferred approach to learn about developmental regulation. The generation of mutant mouse lines however is slow and expensive. One powerful method to introduce specific genetic changes in developing neurons of the mammalian brain is in utero electroporation. Essentially, the technique consists of transfecting DNA into the embryonic brain neuroepithelium by means of electric pulses, then allowing the embryo to survive for a certain period of time, collect the brain and examine them for possible novel, informative phenotypes. In this way, the experimenter can test hypotheses almost immediately with....

Protocol

1. Preparation of DNA and Glass Micropipettes for Injection

1. Good quality glass micropipettes are essential to reduce initial high abortion rate due to loss of amniotic fluid. The procedure to pull glass micropipettes has been well documented^13,18,25. Use 1.2 mm diameter capillaries pulled in a conventional Sutter P-97 device with the settings P=500; Heat=300; Pull=40; Velocity=50; Time=50. Fit the puller with 3 mm "trough" filaments (Sutter Instrument FT330B). The 2 mm size filaments have yielded for us less satisfactory results. On the other hand, beveling of the micropipette tips does not seem to improve results for us.
2. Dissolve puri....

Results

Most hypothalamus neurons are born between E11.5 to E15.2, according to birth-dating analysis in the rat²⁶ translated into the somewhat shorter mouse development^27,28. The peak of hypothalamic neurogenesis is reached at E12.5^29-31. Accordingly, at the transfection age chosen for the present study (E12.5), a large proportion of hypothalamic neurons can be labeled at any given rostro-caudal level.

Analysis at E18.5 on thick vibratome-type sections (Figur.......

Discussion

About the anesthesia: Since in utero electroporation into the hypothalamus can be technically arduous and require longer narcosis times, we prefer to induce and maintain anesthesia through administration of a mixture of oxygen and isoflurane. In our experience, animals can remain suitably anaesthetized in this way for periods of up to one hour at least, the recovery of the mother is very fast, and embryo survival improved. Other approaches to anesthesia are also available. The most simple procedure consis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			REAGENTS
Acepromazine	Sanofi GmbH		anesthetic
Isoflurane	Baxter	HDG9623	anesthetic
Ketamin	Pharma GmbH		anesthetic
Fast Green	Fluka	44715
Rimadyl	Pfizer		non-steroidal anti-inflammatory
Braunoderm	Braun	3887138	povidone-iodine
Phosphate Buffer Saline PBS	Gibco	14190
Temgesic (buprenorphine)	Essex Pharma		opioid analgesic
Eye Ointment	Pan-Ophtal	7136926
Xylazine	Bayer
			EQUIPMENT
Anaesthetic Device Komesaroff Mark-5	Medical Developments Australia	ACN 004 903 682
Capillary puller P-97	Sutter Instrument Co.	P-97
Compresstome	Precisionary Instr.	VF-300	Vibratome-type device
Confocal Microscope	Zeiss	LSM700
Cryostat	Leica	CM3050S
Electroporator	Nepa Gene Co. Ltd.	CUY21EDIT
Electrode 1	Nepa Gene Co. Ltd.	CUY550-10	Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.
Electrode 2	Nepa Gene Co. Ltd.	CUY700P4L	Cover Round Platinum Plate 4 mm diameter
Fiberoptic cold light source	Leica	KL2500 LCD
Glass capillaries	Harvard Apparatus	GC120T-15	1. 2 mm O.D. x 0. 94 mm I.D.
Glass bead sterilizer	Fine Science Tools	FST250
Heating pad	Harvard Apparatus	py872-5272
Injection device	World Precision Instruments	Pneumatic Pico Pump PV820
Suture Thread Coated Vicryl	Ethicon	V4914	Peritoneal Suture
Suture Thr. Supramid	Serag Wiessner	TO07171L	Skin Suture