Summary

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral septal Nucleus- og striatum-specifikke<em> In Utero</em> Elektroporation i C57BL / 6 mus

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

Siden den første beskrivelse i 2001 af tre uafhængige grupper 1-3 i n utero elektroporation er blevet en udbredt standard værktøj til analyse af genekspression i gnavere centralnervesystemet. I forhold til generering af knockout-mus, der er, til trods for løbende at forbedre teknikker, stadig tid og penge tidskrævende, de i utero elektroporation appellerer på grund af sin enkelhed. Så i livmoderen elektroporation muliggør hurtig og effektiv GAIN- og tab-af-funktionen undersøgelser 4.

At transficere de cerebrale områder, er opløsningen indeholdende negativt ladet plasmid injiceret i en ventrikel. Under den elektriske impuls, det negativt ladede DNA vandrer mod den positive pol, og derfor den transfekterede region kan vælges blot ved at ændre positionen af ​​den positive pol. Det er ofte blevet påvist, at mange regioner af centralnervesystemet kan være targeted 3,5-8. For eksempel de seneste undersøgelser viser specifikke transfektioner i hippocampus, den piriform cortex eller striatum 9-11. Er dog, at oplysninger om de relevante positioner ofte kun næppe standardiseret og er ikke altid let at overføre til forskellige musestammer.

Transfektion af visse fosterstadier er langt fra trivielt. Mange faktorer skal tages i betragtning, når de vælger opsætningen for specifik in utero elektroporation. Først optimalt at transficere de respektive fosterstadiet, der er behov for viden om de passende spændinger. Højspænding sænke overlevelsesraten, mens lave spændinger reducerer transfektionseffektiviteten 2,3,12. Også størrelsen af elektroden padle spiller en afgørende rolle, fordi brugen af elektrodematerialer paddles, der er for store resultater i reduceret specificitet eller kan forårsage dødsfald som følge af påvirkning af hjerterytmen 4,12,13. Den anvendtespænding og størrelsen og positionen af ​​elektroden paddle er de vigtigste funktioner til at overveje, men der er også andre faktorer, der påvirker resultatet af elektroporation, som den anvendte mængde DNA-opløsning.

Vi har udviklet en detaljeret protokol, som muliggør hurtig og effektiv transfektion af forskellige cerebrale områder i C57BL / 6 mus 12. I denne protokol detaljeret information om spændinger skal anvendes, og størrelsen af ​​elektroden paddle for øget specificitet er tilvejebragt. Endvidere kan information om ventriklen fyldes sammen med anbefalinger til mængden af ​​plasmidopløsning og positionen af ​​elektroden tilføres. Angivelsen af detaljerede position oplysninger i en kort og den videre visualisering af disse positioner muliggør ligetil specifik og effektiv i livmoderen elektroporation af retrospenialis cortex, den motoriske hjernebark, somatosensoriske cortex, den piriform cortex t,han Cornu ammonis 1-3, gyrus dentatus, striatum, den laterale septum nucleus, thalamus og hypothalamus.

Protocol

Etik Statement: Håndteringen af ​​mus og de eksperimentelle procedurer blev gennemført i overensstemmelse med europæiske, nationale og institutionelle retningslinjer for dyr pleje. 1. I Utero Elektroporation Bemærk: I livmoderen elektroporation blev udført som tidligere offentliggjort 12,14. Derfor er fremgangsmåden kun beskrevet kort i det følgende (figur 1). Forberedelser …

Representative Results

Figur 2 viser eksempler på specifikke in utero elektroporation af regionerne udvikle sig til retrospenialis cortex, den motoriske hjernebark, somatosensoriske cortex i den piriform cortex, Cornu ammonis 1-3, gyrus dentatus, striatum, den laterale septum nucleus , thalamus og hypothalamus. Resultaterne af transfektioner vises ved siden af den anbefalede vinkel (figur 2). For bedre visualisering af vinklerne in vivo position elektroden (0,5 mm) er også vist på et fost…

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…

Disclosures

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

  1. Fukuchi-Shimogori, T., Grove, E. A. Neocortex patterning by the secreted signaling molecule FGF8. Science. 294 (5544), 1071-1074 (2001).
  2. Saito, T., Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol. 240 (1), 237-246 (2001).
  3. Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex. 神经科学. 103 (4), 865-872 (2001).
  4. LoTurco, J., Manent, J. B., Sidiqi, F. New and improved tools for in utero electroporation studies of developing cerebral cortex. Cereb Cortex. 19 (July), i120-i125 (1991).
  5. Nakahira, E., Yuasa, S. Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol. 483 (3), 329-340 (2005).
  6. Navarro-Quiroga, I., Chittajallu, R., Gallo, V., Haydar, T. F. Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation. J Neurosci. 27 (19), 5007-5011 (2007).
  7. Borrell, V., Yoshimura, Y., Callaway, E. M. Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J Neurosci Methods. 143 (2), 151-158 (2005).
  8. Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. M. Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders. FNMOL. 4 (November), 37 (2011).
  9. Tomita, K., Kubo, K. I., Ishii, K., Nakajima, K. Disrupted-in-schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development. Hu Mol Gen. 20 (14), 2834-2845 (2011).
  10. Niwa, M., Kamiya, A., et al. Knockdown of DISC1 by In Utero Gene Transfer Disturbs Postnatal Dopaminergic Maturation in the Frontal Cortex and Leads to Adult Behavioral Deficits. Neuron. 65 (4), 480-489 (2010).
  11. Nakahira, E., Kagawa, T., Shimizu, T., Goulding, M. D., Ikenaka, K. Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes. Dev Biol. 291 (1), 123-131 (2006).
  12. Baumgart, J., Grebe, N. C57BL/6-specific conditions for efficient in utero electroporation of the central nervous system. J Neurosci Methods. 240, 116-124 (2015).
  13. Saito, T. In vivo electroporation in the embryonic mouse central nervous system. Nat Protc. 1 (3), 1552-1558 (2006).
  14. Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. Mouse in utero electroporation: controlled spatiotemporal gene transfection. JoVE. (54), (2011).
  15. Guedel, A. E. . Inhalation anesthesia: a fundamental guide. , (1937).
  16. Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. JoVE. (65), e3564 (2012).
  17. Mizutani, K., Saito, T. Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex. Development. 132 (6), 1295-1304 (2005).
  18. Tabata, H., Nakajima, K. Labeling embryonic mouse central nervous system cells by in utero electroporation. Dev, Growth & Differ. 50 (6), 507-511 (2008).

Play Video

Cite This Article
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).

View Video