移植Dkk-1浸润磁珠到鸡胚神经管

Almudena  Martinez-Ferre; Maria  Navarro-Garberí; Carlos  Bueno; Salvador  Martinez

doi:10.21769/BioProtoc.963

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Peer-reviewed

Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos

移植Dkk-1浸润磁珠到鸡胚神经管

AM Almudena Martinez-Ferre

MN Maria Navarro-Garberí

CB Carlos Bueno

SM Salvador Martinez email

发布: 2013年11月05日第3卷第21期 DOI: 10.21769/BioProtoc.963 浏览次数: 12439

评审: Xuecai 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.

Feb 2013

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Abstract

Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise developmental stages activating or blocking different signaling pathways. Here, we describe the manipulation of Wnt signaling pathway using Dkk-1-soaked micro-beads, implanted in ovo in the anterior part of the developing neural tube of chicken embryos; specifically at the prospective zona limitans intrathalamica at stage HH10.

Keywords: Experimental embryology (实验胚胎学)

Neutral tube (神经管)

Wnt signaling regulation (Wnt信号通路的调控)

Microbeads implant (微种植体)

Ni vivo experimentos (镍体内experimentos)

Materials and Reagents

Fertilized chick (Gallus gallus)
Recombinant mouse Dkk-1 (R&D Systems, catalog number: 1765-DK )
Heparin Acrylic beads (Sigma-Aldrich, catalog number: H5263 )
Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A2153 )
Pelikan Drawing Ink Black
Silikon Peroxid (IDEX Health & Science, catalog number: SC0083ST )
Tungsten wire (0.380 mm, 10 FT) (World Precision Instruments, catalog number: TGW1510 )
Grid with concentric circles (NE42-21 mm) (Graticules LTD, Tonbridge Kent)
Penicillin-Streptomycin (10,000 U/ml) (Gibco, catalog number: 15140-122 )
NaCl (Sigma-Aldrich, catalog number: S-3014 )
KCl (Sigma-Aldrich, catalog number: P-9541 )
CaCl₂^.2H₂O (Prolabo, catalog number: 22317297 )
NaH₂PO₄^.2H₂O (Scharlab, catalog number: SO0334 )
MgCl₂^.6H₂O (Prolabo, catalog number: 27778293 )
D(+) glucose anhydre (Prolabo, catalog number: 24370294 )
10x Phosphate-buffer saline (PBS) (see Recipes)
4% Paraformaldehyde (PFA) from 20% PFA (see Recipes)
PBS/0.1% BSA (see Recipes)
Tyrodes buffer (see Recipes)
0.5% Bicarbonate (see Recipes)
0.5% Glucose (see Recipes)
Tyrodes supplemented (see Recipes)

Equipment

37 °C, 5% CO₂ forced-air incubator (Novital, model: Covatutto 120-4V )
Dissecting microscope (Leica Microsystems, model: MS5 )
Butane burner (Butsir)
Glass micropipet (homemade)
Pasteur pipets (Deltalab, catalog number: 200001 )
Silicon tube (IDEX Health & Science, catalog number: SC0083ST )

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:

Martinez-Ferre, A., Navarro-Garberí, M., Bueno, C. and Martinez, S. (2013). Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos. Bio-protocol 3(21): e963. DOI: 10.21769/BioProtoc.963.
Martinez-Ferre, A., Navarro-Garberi, M., Bueno, C. and Martinez, S. (2013). Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate. J Neurosci 33(9): 3967-3980.