Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin
再生斑马鱼鳍中间充质细胞（包括芽基）的移植
发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2109 浏览次数: 9097
评审: Michelle GoodyAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2016
Abstract
Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.
Keywords: Zebrafish (斑马鱼)
Fin regeneration (鳍再生)
Blastema (芽基)
Transplantation (移植)
Cell lineage (细胞谱系)
Background
Cell transplantation studies are routinely performed during the early embryonic stage in animal models such as fish, amphibians and mammals, but targeting the transplanted cells to specific tissues has been difficult. Blastema transplantation developed in our studies is an efficient method for introducing mesenchymal donor cells into host fin ray. It enables us to track cell fate and/or manipulate cell signaling such as fibroblast growth factor (Fgf) during fish fin regeneration. Actually, in our recently published work, we transplanted blastema cells, which carried the hsp70l:dominant-negative fgf receptor and the β-actin:dsRed2 transgenes, into a wild-type blastema region and mosaically inhibited Fgf signaling in a subset of fin ray mesenchymal cells (Shibata et al., 2016). This method is applicable for analyzing other cell signals and for tracking cell fate by live cell imaging.
Materials and Reagents
- 50 ml disposable syringe (Terumo, catalog number: SS-50ESZ )
- Stainless steel surgical blade, No.10 (FEATHER Safety Razor, catalog number: No. 10 )
- Plastic dish (9 cm diameter) (As One, catalog number: GD90-15 )
- Glass capillary (1 x 90 mm, without filament) (Narishige, catalog number: G-1 )
- Dissection needle: This is made by attaching 30 G needle (BD, catalog number: 305106 ) to a P1000 pipette chip (BM Equipment, catalog number: BIO1000RF ) whose tip is truncated (Figure 1)
Note: A handmade tool for removing the wound epidermis from the regenerate and for dissecting the blastema. This needle is different from that for transplantation (see Procedure B).
Figure 1. Dissection needle - Syringe filter unit (0.22 µm) (EMD Millipore, catalog number: SLGV033RB )
- Zebrafish transplantation donor strain: Tg(Olactb:loxP-dsred2-loxP-egfp), which constitutively expresses the DsRed2 ubiquitously (Yoshinari et al., 2012). For simplicity, we refer the line as Tg(β-actin:dsred2) in this manuscript
- Zebrafish transplantation host: a wild-type strain, which has been maintained in our facility by inbred breeding
- Agarose (Nacalai Tesque, catalog number: 01028-85 )
- Tricaine (3-aminobenzoic acid ethyl ester) (Sigma-Aldrich, catalog number: A5040 )
- Fetal bovine serum (FBS) (any brand can be used)
- Leibowitz’s L-15 medium (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 41300070 )
- 1 M Tris-HCl (pH 9)
- L-15 medium (see Recipes)
- 20x tricaine stock solution (see Recipes)
Equipment
- Microwave oven
- Micropipette puller (Sutter Instrument, model: P-97 )
- Forceps
- Glass dish (5 cm diameter)
- Surgical blade handle (FEATHER Safety Razor, catalog number: No. 3 )
- Microforge (Narishige, model: MF-900 )
- Three-dimensional coarse manual manipulator (Narishhige, model: M-152 ) installed on a magnet stand
- P20 micropipette
- Fluorescence stereomicroscope
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Shibata, E., Ando, K. and Kawakami, A. (2017). Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin. Bio-protocol 7(2): e2109. DOI: 10.21769/BioProtoc.2109.
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分类
发育生物学 > 细胞生长和命运决定 > 再生
干细胞 > 多能干细胞 > 芽基细胞
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