Summary

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Published: January 18, 2016
doi:

Summary

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Abstract

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.

Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Introduction

Since the first description in 2001 by three independent groups 1-3 in utero electroporation has become a widely used standard tool for analyzing gene expression in the rodent central nervous system. Compared to the generation of knockout mice, which is, despite continuously improving techniques, still time and money consuming, the in utero electroporation appeals due to its simplicity. So, in utero electroporation enables fast and efficient gain- and loss-of-function studies 4.

To transfect the cerebral regions, the solution containing the negatively charged plasmid is injected into a ventricle. During the electric pulse, the negatively charged DNA migrates towards the positive pole and therefore the transfected region can be selected simply by altering the position of the positive pole. It has frequently been shown that numerous regions of the central nervous system can be targeted 3,5-8. For instance, recent studies show specific transfections of the hippocampus, the piriform cortex or the striatum 9-11. However, the information about the appropriate positions are often only scarcely standardized and are not always easy to transfer to different mouse strains.

Transfection of certain embryonic stages is far from trivial. Many influencing factors must be taken into consideration when choosing the set-up for specific in utero electroporation. First, to optimally transfect the respective embryonic stages, knowledge about the appropriate voltages is needed. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Also the size of the electrode paddle plays a crucial role, because the use of electrode paddles that are too large results in reduced specificity or can cause death due to affection of the heart rhythm 4,12,13. The applied voltage and the size and the position of the electrode paddle are the most important features to consider, but there are also further factors influencing the outcome of the electroporation, like the applied amount of DNA-solution.

We have developed a detailed protocol which enables fast and efficient transfection of various cerebral regions of the C57BL/6 mouse 12. In this protocol detailed information about the voltages to be used and the size of the electrode paddle for enhanced specificity is provided. Further, information about the ventricle to be filled along with recommendations for the amount of plasmid solution and the position of the electrode is supplied. The indication of the detailed position information in a map and the further visualization of these positions enables straightforward specific and efficient in utero electroporation of the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus.

Protocol

Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care. 1. In Utero Electroporation Note: In utero electroporation was performed as previously published12,14. Therefore, the method is only described briefly in the following (Figure 1). Preparations Prepare Fast Green colored endoto…

Representative Results

Figure 2, shows examples for the specific in utero electroporation of the regions developing into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. The results of the transfections are shown next to the recommended angle (Figure 2). For better visualization of the angles in vivo the position of the electrode (0.5…

Discussion

This protocol describes in detail how to transfect the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Materials

EndoFree Plasmid Maxi Kit QIAGEN 12362
Fast Green Roth 0301.1
pCAGGS Addgene
borosilicate glass capillaries (0.8-0.9 mm diameter) Wold Precision Instrument Inc. 1B100F-4
Isoflurane (Forene) Abbott PZN 4831850
Carprofen (Rimadyl) Pfizer GmbH approval number: 400684.00.00
eye ointment (Bepanthen Augen und Nasensalbe) Bayer  PZN 01578681
0.9% benzyl alcohol 0.9% saline solution Pharmacy
of the University Medical Center Mainz
gauze (ES-Kompressen) Hartmann 407835
sterile 5-0 Perma-Hand Silk Suture Ethicon Johnson & Johnson K890H
ring forceps 1/ 1.5 mm Fine Science Tools 11101-09
ring forceps 4.8/ 6 mm Fine Science Tools 11106-09
ring forceps 2.2/ 3 mm Fine Science Tools 11103-09
Adson Forceps-Serrated Straight 12 cm Fine Science Tools 1106-12
IrisScissors-Delicate Straight-Sharp/Blunt 10 cm Fine Science Tools 14028-10
Mayo-Stille Scissors-Straight 15 cm Fine Science Tools 14012-15
Dumont #5 Forceps-Inox Fine Science Tools 11251-20
Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm Fine Science Tools 12565-14
Elektroporator CUY21 SC  Nepa Gene Co.
FST 250 Hot Bead Sterilizer Fine Science Tools 18000-45
Microgrinder EG-44 Narishige
P-97 Micropette Puller Sutter Instrument Company P-97
Platinum electrodes 650P 0.5 mm Nepagene CUY650P0.5
Platinum electrodes 650P 3 mm Nepagene CUY650P3
Platinum electrodes 650P 5 mm Nepagene CUY650P5
Platinum electrodes 650P 10 mm Nepagene CUY650P10
Anesthesia system Rothacher-Medical GmbH CV-30511-3 Vapor 19.3
Heating plate Rothacher-Medical GmbH HP-1M
Temperature Controller 220V AC Rothacher-Medical GmbH TCAT-2LV

References

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Baumgart, J., Baumgart, N. Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse. J. Vis. Exp. (107), e53303, doi:10.3791/53303 (2016).

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