Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

Summary

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Abstract

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

Introduction

As a well-accepted model organism, zebrafish (Danio rerio) is widely used in scientific research on development, regeneration, tumorigenesis, infection and immunity, etc^1,2,3,4,5. Zebrafish are especially suitable for in vivo live imaging when treated with PTU (1-phenyl 2-thiourea), a toxic chemical compound that inhibits melanogenesis and makes zebrafish larvae transparent for a relatively long period of time⁶. Genetically modified transparent zebrafish strains, su....

Protocol

All zebrafish husbandry and experiments were reviewed and approved by the Laboratory Animal Management and Ethics Committee of Xiamen University and were in strict accordance with good animal practice as defined by Xiamen University Laboratory Animal Center. The study complied with all relevant ethical regulations for animal use.

NOTE: All adult and larval zebrafish were maintained following standard protocols at 28.5 °C with a 13/11 h light/dark cycle in the zebrafish aquarium system of .......

Discussion

To conduct this experiment smoothly and successfully, one should pay more attention to some important aspects. The optimal zebrafish developmental stage for injection should be the one-cell stage. Completing injection within this stage can ensure that all cells produced through subsequent division contain the necessary components for KI, thereby increasing the probability of simultaneous KI occurrence in all cells and improving the overall success rate of KI. Lowering the ambient temperature would slow down zebrafish emb.......

Materials

Name	Company	Catalog Number	Comments
1000 µL  tips	Any brand	N/A	For all manipulations
1.5 mL tubes	Any brand	N/A	For storage and centrifugation of various solutions
1.5 mL tubes (RNase-free)	Any brand	N/A	For storage and centrifugation of RNase-free solutions
10 µL tips	Any brand	N/A	For all manipulations
10 µL tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
1000 µL  tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
1-phenyl 2-thiourea (PTU)	Sigma	P7629	To inhibit melanogenesis and keep the larvae transparent
200 µL tips	Any brand	N/A	For all manipulations
200 µL tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
2x Taq Plus Master Mix II (Dye Plus)	Vazyme	P213-03	PCR for sgRNA efficiency tests
35-mm dishes	Corning	430165	For imaging
6-well plates	Any brand	N/A	For rearing zebrafish individually and temporarily
Acc65I	NEB	R0599S	For enzymatic digestion
Acetic acid	Sigma	695092	For TAE buffer preparation
Agar	Biofroxx	8211GR500	For LB plates
Ampicillin, sodium salt	Sangon	A610028	For LB plates
CaCl2	Sigma	C5080	For embryo buffer preparation
Capillary tubes	Harvard Apparatus	30-0016	For geneating needles for injection
Capillary tubes	Any brand	ID (inside diameter) = 0.1 mm	For quantify a 1-nL droplet
CRISPRscan	CRISPRscan	http://www.crisprscan.org/	For sgRNA design
DH5α	TIANGEN	CB101	For transformation
EDTA	Sigma	E6758	For lysis buffer and TAE buffer preparation
Ensembl	Ensembl	https://asia.ensembl.org/Danio_rerio/Info/Index	To find gene information
Ethanol	HUSHI	64-17-5	For DNA purification and extraction
Exonuclease III	Takara	2170A	LIC
Gas-powered micro-injector	Warner Instruments	PLI-100A	For microinjection
Gel electrophoresis system	WIX	WIX-EP3000	For gel electrophoresis
Gloves	Any brand	N/A	For all manipulations
Glucose	sgdbio	10010518	For LB plates
Glycerol	Sangon	A501745	For LB plates
gRNA-pMD19-T	China Zebrafish Resource Center (CZRC)	CZP3	sgRNA plasmid containing sgRNA scaffold
KCl	Sigma	P5405	For embryo buffer preparation
Lithium chloride precipitation solution	Thermofisher	AM9480	Precipitation of sgRNAs, RNase-free
Low melting point agarose	Sangon	A600015	For preparation of 4% agarose gels
Magnesium sulfate	sgdbio	20025118	For LB plates
Metal bath	Any brand	N/A	For constant temperature incubation
Methylene blue	Sigma	M9140	For embryo buffer preparation
Microinection mold	Homemade	N/A	For generating grooves to hold embryos during microinjection
Microloader, tip for filling Femtotips and other glass microcapillaries, sterile, 0.5 – 20 µL, 100 mm, light gray, 192 pcs. (2 racks × 96 pcs.)	Eppendorf	5242956003	To Load the microinjection solution into the injection needle, RNase-free
Micropipette puller	Sutter Instrument	P-97	For generating needles for injection
Micropipettes	Eppendorf	N/A	For all manipulations
Microwave	Any brand	N/A	For casting injection plate and agarose gels
MinElute PCR Purification Kit (50)	QIAGEN	28004	To purify PCR products and digestion products, RNase-free
mMESSAGE mMACHINE T3 Transcription Kit	Invitrogen	AM1348	In-vitro transcription of Cas9 mRNA, RNase-free
N2	Any brand	N/A	For microinjection
NaCl	Sigma	S5886	For embryo buffer and lysis buffer preparation
NaHCO3	Sigma	S5761	For embryo buffer preparation
NaOH	Sangon	A100173	For TAE buffer preparation
Nuclease-free water	Thermofisher	R0581	RNA related manipulations, RNase-free
PciI	NEB	R0655S	For enzymatic digestion
PCR machine	Any brand	N/A	PCR amplification
PCR strip tubes	Any brand	N/A	For PCR
PCR tubes	FEITONGKANG	blp-200	For PCR
Petri dish	Any brand	N/A	For rearing zebrafish larvae etc.
Phenol red	Sigma	P0290	For microinjection, RNase-free
Pipette holder	Warner Instruments	PLI-PH1	For microinjection
PrimeStar (Premix)	Takara	DR040A	For molecular cloning
Proteinase K	Merck	539480	For genomic DNA extraction
pT3TS (T3:zCas9-UTRglobin)	China Zebrafish Resource Center (CZRC)	CZP11	Cas9 plasmid template
Refrigerated centrifuge	Any brand	N/A	For RNA related centrifugation
Scale	Sartorius	BCE124-1CCN	For casting injection plate and agarose gels
Scissors	Any brand	N/A	For clipping adult fish tails
SDS	Sangon	A600485	For lysis buffer preparation
Small brushes	Any brand	N/A	For gently moving embryos on microinjection plates
Spectrophotometer	Thermofisher	NanoDrop 2000	For DNA/RNA concentration measurements
Stage micrometer	Any brand	DIV (Division) = 0.01 mm	For quantify a 1-nL droplet
Stereo fluorescence microscope	Olympus	SZX16	For observation of gene expression
Stereo microscope	Motic	SMZ-168	For observation and microinjection
T7 RiboMAX Express Large Scale RNA Production System	Promega	P1320	In-vitro transcription of sgRNAs, RNase-free
Temperature-controlled incubator	Any brand	N/A	For embryo incubation
TIDE	TIDE	https://tide.nki.nl/	For sgRNA efficiency analysis
Tricaine methanesulfonate	Sigma	A5040	For tricaine solution preparation
Tris	Sangon	A600194	For tricaine solution, lysis buffer and TAE buffer preparation
Tryptone	OXOID	LP0042	For LB plates
Tweezers	Any brand	N/A	For cutting injection needles
XbaI	NEB	R0145S	For enzymatic digestion
Yeast extract	OXOID	LP0021	For LB plates
Zebrafish aquarium system	ESEN	ESEN-AW-S1	For rearing adult fish
Zebrafish mating tanks (with a divider)	Any brand	N/A	For mating of the fish
Zeiss LSM 900+Airyscan2	ZEISS	Zeiss LSM900	For confocal imaging of the fluorescence-labeled fish and data analysis
ZFIN website	ZFIN	http://zfin.org/	To find gene information