子宫内电穿孔法检测小鼠小脑蒲肯野细胞

Yukari H. Takeo

doi:10.21769/BioProtoc.1835

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Peer-reviewed

In utero Electroporation of Mouse Cerebellar Purkinje Cells

子宫内电穿孔法检测小鼠小脑蒲肯野细胞

Yukari H. Takeo email

发布: 2016年06月05日第6卷第11期 DOI: 10.21769/BioProtoc.1835 浏览次数: 15535

评审: Oneil G. BhalalaEmmanuelle BerretXuecai Ge

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Sep 2015

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Abstract

In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.

Keywords: Purkinje cell (浦肯野细胞)

In utero electroporation (子宫穿孔)

Cerebellum (小脑)

Materials and Reagents

Glass capillary [(Sutter Instrument Company, catalog number: BF100-50-10 ) for microinjector or (World Precision Instruments, catalog number: 1B150F-3 ) for mouth pipette]
Sterile gauze (KAWAMOTO CORPORATION, catalog number: 7164 )
Surgical tape (3M, catalog number: 1527SP-0 )
Surgical scalpel blade (Swann Morton, model: No.11 )
Suture needle (Alfresa pharma, catalog number: HT1605 NA75-KF2 )
E11.5 (or E10~12) pregnant mouse
QIAFilter plasmid maxi kit (QIAGEN, catalog number: 12263 )
Fast green (Sigma-Aldrich, catalog number: F7258 )
Sodium pentobarbital (Somnopentyl) (KYORITSU SEIYAKU CORPORATION, catalog number: 4992945014418 )
Ethanol
Ritodrine hydrochloride (Sigma-Aldrich, catalog number: R0758 )
(Optional) EEG conductive paste (NIHON KOHDEN CORPORATION, catalog number: Z181JE )
HEPES (Sigma-Aldrich, catalog number: H4034 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Disodium hydrogenphosphate 12-water (Na₂HPO₄･12H₂O) (Wako Pure Chemical Industries, catalog number: 196-02835 )
Potassium dihydrogen phosphate (KH₂PO₄) (Wako Pure Chemical Industries, catalog number: 169-04245 )
HEPES-buffered saline (HBS) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)

Equipment

Micropipette puller (Sutter Instrument Company, model: P-87 )
Microscope with 40x objective (Nikon Instruments Inc., model: Eclipse E100 )
Scissors, fine forceps and ring forceps
Square wave electroporator (Nepa Gene Co., model: CUY21SC )
Tweezers-type electrodes (Nepa Gene Co., model: CUY650P3 )
Microinjector (Eppendorf AG, model: 5242 ) or mouth pipette
LED light source with flexible light guide (Kenko Tokina, model: KTX-20L )
Reflex 7 mm wound clip applier (Cellpoint Scientific Inc., catalog number: 204-1000 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Takeo, Y. H. (2016). In utero Electroporation of Mouse Cerebellar Purkinje Cells. Bio-protocol 6(11): e1835. DOI: 10.21769/BioProtoc.1835.
Takeo, Y. H., Kakegawa, W., Miura, E. and Yuzaki, M. (2015). RORalpha regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo. J Neurosci 35(36): 12518-12534.