Name: fluorescence labeling to visualize low-expressed proteins in zebrafish
Uploaded: 2025-01-24T14:40:00.000+00:00
Duration: 9 min 38 s
Description: CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

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

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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 embryonic development18. However, prolonged exposure of embryos to low temperatures increases mortality rates. Therefore, we emphasize that embryos should not be placed directly on ice or cold holders, nor should they be kept at low temperatures for too long. Instead, we maintain a relatively low ambient temperature (16 &#176;C) to slightly slow down the embryonic development, giving us sufficient time for injection, which should be done as quickly as possible. Typically, an injection for about 100 embryos can be done within 30 min. Afterward, the embryos should be promptly transferred into a 28.5 &#176;C constant temperature incubator to ensure their viability to the greatest extent.
The volume of liquid injected into the embryos should be strictly limited to around 1 nL. A lower volume will reduce the efficiency of KI, while a higher volume will cause embryonic death. Based on our observation, healthy zebrafish embryos can stand an injection volume of 1 nL without affecting the survival and development of the embryos. Therefore, we choose 1 nL to ensure high KI efficiency and survival rates.
Pay special attention to RNase-free operations to avoid experimental failures due to RNA degradation. Measures include but are not limited to cleaning bench surfaces, centrifuges, and other related equipment with 0.5% SDS followed by 3% H2O2; wearing a lab coat and gloves; treating home-made reagents, such as water, with DEPC (0.5 mL/L DEPC in water, incubate for 2 h and then autoclave for 45 min); using RNase-free tubes and filtered tips; achieving RNase-free status of the injection mix by ensuring that all components and manipulations are RNase-free; using a kit containing RNase-free reagents to purify plasmids and DNA templates or to do in vitro transcription and using a spectrophotometer to detect any presence of contaminated proteins; long-term storing RNAs in ethanol at -80 &#176;C(Prior to use, centrifuge to pellet the RNA, air dry and then resuspend in RNase-free water; When in use, place the RNAs on ice whenever possible); further ensuring the stability of RNAs by lowering the ambient temperature during injection and improving the injection skills to decrease the manipulation time.
This protocol is particularly suitable for studies on genes expressed within 96 hpf when the zebrafish larvae remain relatively transparent. Modifications are required when one needs to do the imaging during adulthood, for example, by using zebrafish with a casper or crystal background rather than the WT T&#252;. But it should be noted that the casper is a zebrafish line that is doubly mutant for both nacre and roy7 and the crystal is an optically clear zebrafish mutant combining different viable pigmentation mutations8. Therefore, these two zebrafish lines are useful for imaging in adult fish but are not suitable tools if the purpose is to study pigmentation-related processes.
The described method was successfully applied recently13 to label and visualize multiple members of the connexin family and genes crucial for embryonic development. Fluorescent labeling was proven to maintain the endogenous expression of the target gene while signal amplification by qPCR, which is a suggested validation test when applying this protocol to other genes. However, it should be noted that this protocol is designed for C-terminal tagging and would incorporate an artificial 3&#39; UTR and a poly-A sequence. The possibility that the endogenous expression level or the subcellular localization of the protein might be disturbed cannot be excluded. Therefore, to verify the protein expression and localization, researchers can compare the fluorescence patterns with published data, such as data on ZFIN (https://zfin.org/action/expression/search), or do an in-situ hybridization as a control experiment.
When a high-efficiency sgRNA is unavailable, a sgRNA with a knockout efficiency of about 30% is also acceptable. It happens that for some genes, it may not be possible to find a high-efficiency sgRNA for the target region. In practice, the lowest efficiency of sgRNAs we have used for successful tagging is 30%, which will undoubtedly increase the workload of microinjection and subsequent genotyping and screening.
Off-target is an issue for all gene-editing methods, and currently, we are still relying on the predictions of online software. sgRNAs with a predicted low off-target possibility should be chosen. To validate the efficiency and accuracy of the KI, previously13, we used the published Cyclic Digestion and Ligation Mediated PCR (CDL-PCR) method19, which is also recommended when using this protocol.
Statistical data during KI should be collected and analyzed, including the editing efficiency, fluorescence levels, survival rate, and the proportion of effective F1 progeny.
If a large number of embryos die after injection, possible reasons include incorrect injection volume, which requires re-quantification; poor condition of the embryos, making it necessary to train the parental fish two to three times before injection; embryos not kept moist during the injection; physical damages caused by broken injection needles or unskilled injection; direct contact of embryos to cold objects.
If a successful KI F0 cannot be obtained, possible reasons include low sgRNA efficiency and sgRNA degradation. Too low sgRNA efficiency will require re-screening of sgRNA. Failure to obtain KI F1 from the correct F0 may be because the injection in F0 was not completed at the one-cell stage. Another reason could be that the endogenous gene expression is disturbed, affecting the reproductive system.
If the correct KI strain is obtained but no fluorescence is observed, possible reasons include: (i) The gene selected does not express during the embryonic stage. (ii) The endogenous gene expression is disrupted by editing. (iii) The endogenous gene expression is below detection.
In sum, based on our experience and comparison13, there are several advantages of this method. It is suitable for fluorescence labeling and signal amplification for genes with low expression levels and genes essential for embryonic development. The donor construction process is easy and fast since components except for the CDS are incorporated into the backbone of the donor beforehand, allowing for easily constructing multiple donors with the same tag at the same time. The germline transmission screen workflow set an example of accumulating inheritable KI alleles at the lowest cost of time and effort. To improve the efficiency, the donor was optimized by reducing the number of Cas9/sgRNA binding sites from two to one, shortening the length of the homologous arms to 25 bp, and reversing the sequence of the Cas9/sgRNA binding site from CCN to NGG13.

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><tbody><tr><td>1000 &amp;micro;L&amp;nbsp; 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 &amp;micro;L tips</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>10 &amp;micro;L tips (RNase-free)</td><td>Any brand</td><td>N/A</td><td>For RNase-free manipulations</td></tr><tr><td>1000 &amp;micro;L&amp;nbsp; 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 &amp;micro;L tips</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>200 &amp;micro;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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>&amp;nbsp;http://www.crisprscan.org/</td><td>For sgRNA design</td></tr><tr><td>DH5&amp;alpha;</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>&amp;nbsp;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 &amp;ndash; 20 &amp;micro;L, 100 mm, light gray, 192 pcs. (2 racks &amp;times; 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>N&lt;sub&gt;2&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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&amp;nbsp;</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, inheritable specific fluorescence was observed in muscles of all F1 progeny, indicating the successful GFP labeling of connexin 39.9 (Figure 5A). In consistence with a previous report17, inheritable specific fluorescence was detected in the lens of all F2 progeny, suggesting successful mCherry tagging of connexin 44.1, in the case of which the fluorescence was not observed in F0 but appeared in F2 after screen following our workflow (Figure 5B). Based on these images, we can conclude that connexin 39.9 is highly expressed in muscle fibers and connexin 44.1 is highly expressed in the lens.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67616/67616fig01.jpg" /> 
Figure 1. sgRNA scaffold on the sgRNA plasmid <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67616/67616fig02.jpg" /> 
Figure 2. Illustration of the insert of the donor plasmid: 1, the right homology arm; 2, L-golden site (5&#39;-gggtcaacgacttcctgcacggg-3&#39;, the underlined italic &#39;ggg&#39; represents the PAM); 3, the left homology arm; 4, synonymous mutation sequence. F1, forward primer 1; F2, forward primer 2; R1, reverse primer 1; R2, reverse primer 2. CDS, coding sequence, which will be amplified by PCR from the cDNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67616/67616fig03.jpg" /> 
Figure 3. Illustration of the donor plasmid. 1, the right homology arm; 2, L-golden site; 3, the left homology arm; 4, synonymous mutation sequence; Gal4 gene, encoding the yeast transcription activator protein GAL4; VP64, transcriptional activator; HSF1, transcription activator; Upstream activation sequence (UAS), an enhancer to which GAL4 specifically binds to activate gene transcription; E1b, promoter; eGFP, fluorescent protein; Amp, Ampicillin resistance gene; ori, origin of replication. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67616/67616fig04.jpg" /> 
Figure 4. Illustration of the MMEJ-mediated KI. 1, the right homology arm; 3, the left homology arm; 4, synonymous mutation sequence. All the rest of the elements are the same as those in Figure 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67616/67616fig05.jpg" /> 
Figure 5. Representative imaging results. The fluorescent protein-tagging method described here was used to label (A) connexin 39.9 and (B) connexin 44.1. (A) Inheritable specific fluorescence was observed in the muscles of all F1 progeny. Scale bars: 500 &#181;m. (B) Inheritable specific fluorescence was detected in the lens of all F2 progeny. Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67616/67616fig06.jpg" /> 
Figure 6. Representativejunction PCR results. Correct KI was verified by performing junction PCRs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/67616fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Formulation for buffers used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2. Primer sequences. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3. Reaction mixes used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4. The PCR program used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5. The microinjection mix for knockout. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 5.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6. Sequences of elements in the donor vector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 6.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7. The microinjection mix for KI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Table 7.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1. Donor plasmid sequence of cx39.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Supplementary File 1.dna" target="_blank">Please click here to download this File.</a>
Supplementary File 2. Donor plasmid sequence of cx44.1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Supplementary File 2.dna" target="_blank">Please click here to download this File.</a>
Supplementary File 3. Designed DNA and protein sequences of cx39.9 and cx44.1 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67616/Supplementary File 3.docx" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Genetics

436 Views.  Xiamen University. Dit product is geoptimaliseerd om eiwitten te nivelleren met de fluorescentie-eiwittag en om de locatie en de expressie van het eiwit van een interessant zebravislab te visualiseren. Het is vooral nuttig voor het afbeelden van eiwitten die in een lage overvloed tot expressie worden gebracht. Onze methode vereenvoudigt en versnelt het proces van donoropbouw.Deze componenten, met uitzondering van de CDS, worden vooraf in de backbone opgenomen. De donor w...

Video: Fluorescentielabeling om eiwitten met een lage expressie in zebravissen te visualiseren

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Developmental Biology

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Biology

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

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