An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

摘要

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

摘要

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

引言

Zebrafish is a small tropical hard-bones freshwater fish originated in India. Regarding the gene conservation, zebrafish have a similarity of 87% to the human¹. It can provide us insights on related subjects of human by studying the gene regulation, protein function and cell behavior such as migration, proliferation et.al in zebrafish. Zebrafish embryo can be used to observe the development of early embryos at different stages after inhibiting pigment. Meanwhile, it takes only three months for zebrafish to develop into sexual maturity, then the zebrafish can produce hundreds of eggs every 4 days. Mini-size, simple breeding, strong reproductive ....

方案

NOTE: All the samples were treated by phenylthiourea(PTU) egg water to inhibit pigment.

1. Generation of Tg (fabp10a:il34) Transgenic Constructs and Fish Injection

1. Clone the 2.8 kb fabp10a promoter⁸ and the IL-34 coding regions (ENSDART00000126460.3) of zebrafish into the pTol2 vector to generate the fabp10a-il34 construct. Inject the constructs into one-cell stage Tg (mpeg1: GFP) and WT fish embryos together with the transposase mRNA. Raise the fabp10a-il34 injected WT embryos to adult⁹ and identify the transgenic found....

结果

The steps involved in the protocol of zebrafish are illustrated in Figure 2. First, we generated the pBLK-fabp10a-il34-sv40 construct in which il34 was driven by the fabp10a promoter (Figure 2). The construct was microinjected into one-cell stage Tg (mpeg1: GFP) zebrafish embryos which can label macrophages with GFP and WT embryos which were raised to adults to generate transgenic stable line (.......

讨论

The protocol described here allows us to investigate the function of a chemokine on the behavior of macrophagein vivo and the procedure requires some technical expertise. In summary, there are several critical steps to avoid complications in the protocol: 1) select a suitable transgenic line which shows specific and strong transgenic signal to label the cell of interest; 2) select an appropriate tissue which is accessible for imaging and transgenic gene overexpression; 3) make a sensitive and specific RNA probe; 4) selec.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
抗体
Alexa 488-抗山羊抗体	Invitrogen	A11055
抗地高辛-HRP	珀金埃尔默	NEF832001EA
山羊抗 GFP 抗体	Abcam	ab6658
Reagent
CaCl2· 2H2O	Sigma	21097
花青素 3 Plus 扩增试剂	perkinelmer	NEL745001KT
E2溶液			15 mM 氯化钠 +0.5 mM KCl +1.0 mM MgSO4+150 &微量;M KH2PO4 + 50 µM Na2HPO4 +1.0 mM CaCl2 + 0.7 mM NaHCO3
胎牛血清 （FBS）	Life	10099-133
甲酰胺	钻石	A100314
甘油	Sigma	V900860
肝素钠	Sigma	H3149
杂交缓冲液（HB）			50% 甲酰胺 + 5&次;SSC + 9 mM 柠檬酸钠 + 50 μg/ml 肝素钠 + 500 μg/mL tRNA+ 0.1% Tween20
KCl	Sigma	P5405
KH2PO4	Sigma	P5655
低熔点琼脂糖	Sigma	A9414
甲醇	GHTECH	1.17112.023
亚甲蓝	Sigma	M9140
MgSO4	Sigma	M2643
Na2HPO4	Sigma	S5136
NaCl	Sigma	S5886
NaHCO3	Sigma	S5761
多聚甲醛 （PFA）	Sigma	158127	将 16 g PFA 悬浮在 400 ml 1x PBS 中，在 60 °C 下加热;C 溶解约 30 分钟。该溶液可提前制备并储存在 -4 °C。使用蒙版进行作。
10&次;PBS			14.2 g Na2HPO4+80 g NaCl+2 g KCl + 2.4 g KH2PO4 在 1L ddH2O
苯硫脲 （PTU）	Sigma	P7629
1&次中;Plus 扩增稀释剂	perkinelmer	NEL745001KT
蛋白酶 K	发酵剂	E00492
20&次;盐水柠檬酸钠 （SSC）			175.3 g NaCl + 88.2 g 柠檬酸钠溶于 1 L ddH2O 中，PH 7.0
柠檬酸钠	Sigma	A5040
三卡因	Sigma	E10521
tRNA	Sigma	R6625
吐温20	Sigma	P2287
质粒
pBLK-fabp10a-il34-sv40			用于 Tg （fab10a：il34）转基因品系生成
pBSK-il34			用于 il34 探针制备
Fish
Tg （mpeg1： GFP）			用 GFP
Tg （fabp10a： DsRed）标记巨噬细胞			DsRed
Tg （fab10a：il34） 标记肝细胞			在肝细胞中过表达 IL-34