An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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....

Results

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 (.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibody
Alexa 488-Anti-Goat antibody	Invitrogen	A11055
Anti-Digoxigenin-HRP	perkinelmer	NEF832001EA
Goat-Anti-GFP antibody	Abcam	ab6658
Reagent
CaCl2· 2H2O	Sigma	21097
Cyanine 3 Plus Amplification Reagent	perkinelmer	NEL745001KT
E2 solution			15 mM  NaCl +0.5 mM KCl +1.0 mM MgSO4+150 µM  KH2PO4 + 50 µM  Na2HPO4 +1.0 mM CaCl2 + 0.7 mM NaHCO3
Fetal Bovine Serum (FBS)	Life	10099-133
Formamide	Diamond	A100314
Glycerol	Sigma	V900860
Heparin sodium	Sigma	H3149
Hybridization buffer(HB)			50% formamide+ 5×SSC+9 mM sodium citrate+50 μg/ml heparin sodium+ 500 μg/mL tRNA+ 0.1% Tween20
KCl	Sigma	P5405
KH2PO4	Sigma	P5655
Low melting agarose	Sigma	A9414
Methanol	GHTECH	1.17112.023
Methylene blue	Sigma	M9140
MgSO4	Sigma	M2643
Na2HPO4	Sigma	S5136
NaCl	Sigma	S5886
NaHCO3	Sigma	S5761
Paraformaldehyde (PFA)	Sigma	158127	Suspend 16 g of PFA in 400 ml of 1x PBS, heat at 60 °C  to dissolve about 30 min. This solution can be prepared in advance and stored at -4 °C. Caution. Manipulate with mask.
10×PBS			14.2 g Na2HPO4+80 g NaCl+2 g KCl+ 2.4 g KH2PO4 in 1L ddH2O
Phenylthiourea (PTU)	Sigma	P7629
1×Plus Amplification Diluent	perkinelmer	NEL745001KT
Proteinase K	Fermentas	E00492
20×Saline sodium citrate(SSC)			175.3 g NaCl+ 88.2 g sodium citrate in 1 L ddH2O, PH 7.0
Sodium citrate	Sigma	A5040
Tricaine	Sigma	E10521
tRNA	Sigma	R6625
Tween20	Sigma	P2287
Plasmid
pBLK-fabp10a-il34-sv40			For Tg (fab10a:il34) transgenic line generation
pBSK-il34			For il34 probe preparation
Fish
Tg (mpeg1: GFP)			Label macrophages with GFP
Tg (fabp10a: DsRed)			Label liver cells with DsRed
Tg (fab10a:il34)			Over-expression IL-34 in liver cells