We thank Lu Zhou (Xiamen University) for proofreading and editing this manuscript. This work was supported by the National Natural Science Foundation of China (grant 82388201 to J.H.; grant 31801158 to Y.Z.), the National Key R&#38;D Program of China (2020YFA0803500 to J.H.), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-062 to J.H.), the Fujian Province Central to Local Science and Technology Development Special Program (2022L3079 to J.H.), and the FuXia-Quan Zi-Chuang District Cooperation Program (3502ZCQXT2022003 to J.H.). The funders had no role in study design, data collection and analysis, publication decisions, or manuscript preparation.

Fluorescent Protein Tagging in Zebrafish Larvae Using CRISPR/Cas9 Knock-In

<ol>
	<li>Brittijn, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53 (5-6), 835-850 (2009).</li><li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends Genet. 29 (11), 611-620 (2013).</li><li>Haraoka, Y., Miyake, M., Ishitani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+imaging+reveals+hidden+oncogenic-normal+cell+communication+during+primary+tumorigenesis.">Zebrafish imaging reveals hidden oncogenic-normal cell communication during primary tumorigenesis.</a> Cell Struct Funct. 48 (1), 113-121 (2023).</li><li>Gomes, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+transparent+zebrafish:+a+refined+method+to+improve+detection+of+gene+expression+during+embryonic+development.">Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.</a> Mar Biotechnol (NY). 3 (6), 522-527 (2001).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Antinucci, P., Hindges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crystal-clear+zebrafish+for+in+vivo+imaging.">A crystal-clear zebrafish for in vivo imaging.</a> Sci Rep. 6, 29490 (2016).</li><li>He, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+ligase+3-dependent+microhomology-mediated+end+joining+repair+of+DNA+double-strand+breaks+in+zebrafish+embryos.">Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.</a> Mutat Res. 780, 86-96 (2015).</li><li>Hisano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+in-frame+integration+of+exogenous+DNA+mediated+by+CRISPR/Cas9+system+in+zebrafish.">Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.</a> Sci Rep. 5, 8841 (2015).</li><li>Luo, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-based+genome+engineering+of+zebrafish+using+a+seamless+integration+strategy.">CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.</a> FASEB J. 32 (9), 5132-5142 (2018).</li><li>Wierson, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeted+integration+directed+by+short+homology+in+zebrafish+and+mammalian+cells.">Efficient targeted integration directed by short homology in zebrafish and mammalian cells.</a> Elife. 9, e53968 (2020).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+tagging+strategies+for+illuminating+expression+profiles+of+genes+with+different+abundance+in+zebrafish.">Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish.</a> Commun Biol. 6 (1), 1300 (2023).</li><li>Li, C., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+independent+cloning+irrespective+of+restriction+site+compatibility.">Ligation independent cloning irrespective of restriction site compatibility.</a> Nucleic Acids Res. 25 (20), 4165-4166 (1997).</li><li>Yu, C., Zhang, Y., Yao, S., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+based+protocol+for+detecting+indel+mutations+induced+by+TALENs+and+CRISPR/Cas9+in+zebrafish.">A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.</a> PLoS One. 9 (6), e98282 (2014).</li><li>Hirata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+39.9+protein+is+necessary+for+coordinated+activation+of+slow-twitch+muscle+and+normal+behavior+in+zebrafish.">Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish.</a> J Biol Chem. 287 (2), 1080-1089 (2012).</li><li>Cason, N., White, T. W., Cheng, S., Goodenough, D. A., Valdimarsson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+expression+analysis,+and+functional+characterization+of+connexin44.1:+a+zebrafish+lens+gap+junction+protein.">Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.</a> Dev Dyn. 221 (2), 238-247 (2001).</li><li>Urushibata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+developmental+speed+in+zebrafish+embryos+using+different+incubation+temperatures.">Control of developmental speed in zebrafish embryos using different incubation temperatures.</a> Zebrafish. 18 (5), 316-325 (2021).</li><li>Yu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+digestion+and+ligation-mediated+PCR+used+for+flanking+sequence+walking.">Cyclic digestion and ligation-mediated PCR used for flanking sequence walking.</a> Sci Rep. 10 (1), 3434 (2020).</li></ol>

All authors declare no competing interests in this paper.

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...

As a well-accepted model organism, zebrafish (Danio rerio) is widely used in scientific research on development, regeneration, tumorigenesis, infection and immunity, etc1,2,3,4,5. 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, such as the casper7 and the crystal8, are also available, allowing for live visualization and lineage tracing till adulthood. Besides these efforts to overcome pigmentation for better live visualization in adult fish, our research also focuses on developing powerful gene editing strategies to make fluorescent protein tagging more efficient and less artificial.
CRISPR/Cas9-mediated microhomology-mediated end joining (MMEJ)-based KI technology is now widely applied in genetic modifications in multiple organisms, including zebrafish, due to its relatively high KI efficiency9,10,11,12. To achieve successful in vivo imaging, two challenges need to be addressed. One is the indels or scars introduced by the process of recombination; the other one is that some target genes are endogenously expressed at a low level, leading to weak signals of the co-expressed fluorescence tag and the failure of in vivo detection. Aiming for efficient and accurate protein tagging and minimizing the interference of endogenous gene expression levels, a recent work reported an optimized strategy combining a fluorescence-signal-amplifying &#34;VH&#34; tagging method and a refined germline screen workflow13, which is more suitable for fluorescence labeling low expressed genes and more efficient than previous methods by optimizing the donor backbone and combining polymerase chain reaction (PCR) and fluorescence verifications to implement germline screen at the lowest cost of time and effort. Here, we present a detailed step-by-step protocol for the reported method, including steps of embryo preparation and zebrafish husbandry, sgRNA design, in vitro transcription of sgRNAs, in vitro transcription of Cas9 mRNA, zebrafish embryo microinjection, in vivo screen for sgRNAs with the highest efficiency, donor plasmid design and construction, KI zebrafish genotyping, KI germline screen, and KI zebrafish imaging and phenotyping. This all-in-one protocol, providing a state-of-the-art fluorescence labeling method, would no doubt promote studies using zebrafish in various scientific fields and benefit readers from different academic backgrounds.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L&#160; tips</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For all manipulations</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For storage and centrifugation of various solutions</td>
		</tr>
		<tr>
			<td>1.5 mL tubes (RNase-free)</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For storage and centrifugation of RNase-free solutions</td>
		</tr>
		<tr>
			<td>10 &#181;L tips</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For all manipulations</td>
		</tr>
		<tr>
			<td>10 &#181;L tips (RNase-free)</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For RNase-free manipulations</td>
		</tr>
		<tr>
			<td>1000 &#181;L&#160; tips (RNase-free)</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For RNase-free manipulations</td>
		</tr>
		<tr>
			<td>1-phenyl 2-thiourea (PTU)</td>
			<td>Sigma</td>
			<td>P7629</td>
			<td>To inhibit melanogenesis and keep the larvae transparent</td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For all manipulations</td>
		</tr>
		<tr>
			<td>200 &#181;L tips (RNase-free)</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For RNase-free manipulations</td>
		</tr>
		<tr>
			<td>2x Taq Plus Master Mix II (Dye Plus)</td>
			<td>Vazyme</td>
			<td>P213-03</td>
			<td>PCR for sgRNA efficiency tests</td>
		</tr>
		<tr>
			<td>35-mm dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For rearing zebrafish individually and temporarily</td>
		</tr>
		<tr>
			<td>Acc65I</td>
			<td>NEB</td>
			<td>R0599S</td>
			<td>For enzymatic digestion</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>695092</td>
			<td>For TAE buffer preparation</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Biofroxx</td>
			<td>8211GR500</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>Ampicillin, sodium salt</td>
			<td>Sangon</td>
			<td>A610028</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td>For embryo buffer preparation</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Harvard Apparatus</td>
			<td>30-0016</td>
			<td>For geneating needles for injection</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Any brand</td>
			<td>ID (inside diameter) = 0.1 mm</td>
			<td>For quantify a 1-nL droplet</td>
		</tr>
		<tr>
			<td>CRISPRscan</td>
			<td>CRISPRscan</td>
			<td>&#160;http://www.crisprscan.org/</td>
			<td>For sgRNA design</td>
		</tr>
		<tr>
			<td>DH5&#945;</td>
			<td>TIANGEN</td>
			<td>CB101</td>
			<td>For transformation</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td>For lysis buffer and TAE buffer preparation</td>
		</tr>
		<tr>
			<td>Ensembl</td>
			<td>Ensembl</td>
			<td>&#160;https://asia.ensembl.org/Danio_rerio/Info/Index</td>
			<td>To find gene information</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>HUSHI</td>
			<td>64-17-5</td>
			<td>For DNA purification and extraction</td>
		</tr>
		<tr>
			<td>Exonuclease III</td>
			<td>Takara</td>
			<td>2170A</td>
			<td>LIC</td>
		</tr>
		<tr>
			<td>Gas-powered micro-injector</td>
			<td>Warner Instruments</td>
			<td>PLI-100A</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Gel electrophoresis system</td>
			<td>WIX</td>
			<td>WIX-EP3000</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For all manipulations</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>sgdbio</td>
			<td>10010518</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sangon</td>
			<td>A501745</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>gRNA-pMD19-T</td>
			<td>China Zebrafish Resource Center (CZRC)</td>
			<td>CZP3</td>
			<td>sgRNA plasmid containing sgRNA scaffold</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>For embryo buffer preparation</td>
		</tr>
		<tr>
			<td>Lithium chloride precipitation solution</td>
			<td>Thermofisher</td>
			<td>AM9480</td>
			<td>Precipitation of sgRNAs, RNase-free</td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sangon</td>
			<td>A600015</td>
			<td>For preparation of 4% agarose gels</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>sgdbio</td>
			<td>20025118</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>Metal bath</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For constant temperature incubation</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M9140</td>
			<td>For embryo buffer preparation</td>
		</tr>
		<tr>
			<td>Microinection mold</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td>For generating grooves to hold embryos during microinjection</td>
		</tr>
		<tr>
			<td>Microloader, tip for filling Femtotips and other glass microcapillaries, sterile, 0.5 &#8211; 20 &#181;L, 100 mm, light gray, 192 pcs. (2 racks &#215; 96 pcs.)</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>To Load the microinjection solution into the injection needle, RNase-free</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>For generating needles for injection</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td>For all manipulations</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For casting injection plate and agarose gels</td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit (50)</td>
			<td>QIAGEN</td>
			<td>28004</td>
			<td>To purify PCR products and digestion products, RNase-free</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE T3 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1348</td>
			<td>In-vitro transcription of Cas9 mRNA, RNase-free</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td>For embryo buffer and lysis buffer preparation</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>For embryo buffer preparation</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sangon</td>
			<td>A100173</td>
			<td>For TAE buffer preparation</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Thermofisher</td>
			<td>R0581</td>
			<td>RNA related manipulations, RNase-free</td>
		</tr>
		<tr>
			<td>PciI</td>
			<td>NEB</td>
			<td>R0655S</td>
			<td>For enzymatic digestion</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>PCR amplification</td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For PCR</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>FEITONGKANG</td>
			<td>blp-200</td>
			<td>For PCR</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For rearing zebrafish larvae etc.</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P0290</td>
			<td>For microinjection, RNase-free</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Warner Instruments</td>
			<td>PLI-PH1</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>PrimeStar (Premix)</td>
			<td>Takara</td>
			<td>DR040A</td>
			<td>For molecular cloning</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Merck</td>
			<td>539480</td>
			<td>For genomic DNA extraction</td>
		</tr>
		<tr>
			<td>pT3TS (T3:zCas9-UTRglobin)</td>
			<td>China Zebrafish Resource Center (CZRC)</td>
			<td>CZP11</td>
			<td>Cas9 plasmid template</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For RNA related centrifugation</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>BCE124-1CCN</td>
			<td>For casting injection plate and agarose gels</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For clipping adult fish tails</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sangon</td>
			<td>A600485</td>
			<td>For lysis buffer preparation</td>
		</tr>
		<tr>
			<td>Small brushes</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For gently moving embryos on microinjection plates</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermofisher</td>
			<td>NanoDrop 2000</td>
			<td>For DNA/RNA concentration measurements</td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>Any brand</td>
			<td>DIV (Division) = 0.01 mm</td>
			<td>For quantify a 1-nL droplet</td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>For observation of gene expression</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Motic</td>
			<td>SMZ-168</td>
			<td>For observation and microinjection</td>
		</tr>
		<tr>
			<td>T7 RiboMAX Express Large Scale RNA Production System</td>
			<td>Promega</td>
			<td>P1320</td>
			<td>In-vitro transcription of sgRNAs, RNase-free</td>
		</tr>
		<tr>
			<td>Temperature-controlled incubator</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For embryo incubation</td>
		</tr>
		<tr>
			<td>TIDE</td>
			<td>TIDE</td>
			<td>https://tide.nki.nl/</td>
			<td>For sgRNA efficiency analysis&#160;</td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td>For tricaine solution preparation</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sangon</td>
			<td>A600194</td>
			<td>For tricaine solution, lysis buffer and TAE buffer preparation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For cutting injection needles</td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>NEB</td>
			<td>R0145S</td>
			<td>For enzymatic digestion</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td>For LB plates</td>
		</tr>
		<tr>
			<td>Zebrafish aquarium system</td>
			<td>ESEN</td>
			<td>ESEN-AW-S1</td>
			<td>For rearing adult fish</td>
		</tr>
		<tr>
			<td>Zebrafish mating tanks (with a divider)</td>
			<td>Any brand</td>
			<td>N/A</td>
			<td>For mating of the fish</td>
		</tr>
		<tr>
			<td>Zeiss LSM 900+Airyscan2</td>
			<td>ZEISS</td>
			<td>Zeiss LSM900</td>
			<td>For confocal imaging of the fluorescence-labeled fish and data analysis</td>
		</tr>
		<tr>
			<td>ZFIN website</td>
			<td>ZFIN</td>
			<td>http://zfin.org/</td>
			<td>To find gene information</td>
		</tr>
	</tbody>
</table>

fluorescence labeling to visualize low-expressed proteins in zebrafish

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.

Gap junctions consisting of polymeric proteins called connexins are essential for the exchange of low-molecular-weight metabolites and ions between contacting cells. The fluorescent protein-tagging method described above was used to label connexin 39.9 (Figure 5A) and connexin 44.1 (Figure 5B), respectively. Correct KI was verified by junction PCRs (Figure 6). In agreement with published data16</s...

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

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.

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

fluorescence-labeling-to-visualize-low-expressed-proteins-in-zebrafish

Research

JoVE Journal

Genetics

243 Views.  Xiamen University. 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.

Video: Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...