www.liebertpub.com
Open in
urlscan Pro
65.156.1.51
Public Scan
URL:
https://www.liebertpub.com/doi/10.1089/zeb.2019.1796
Submission: On June 27 via manual from US — Scanned from DE
Submission: On June 27 via manual from US — Scanned from DE
Form analysis 7 forms found in the DOM
POST /action/doLogin
<form action="/action/doLogin" method="post"><input type="hidden" name="id" value="119162c4-9551-4055-99fe-48538e2570bc">
<input type="hidden" name="redirectUri" value="/doi/10.1089/zeb.2019.1796">
<input type="hidden" name="loginUri" value="/doi/10.1089/zeb.2019.1796">
<input type="hidden" name="popup" value="true">
<div class="input-group">
<div class="label ">
<label for="login">Username</label>
</div>
<input id="login" class="login" type="text" name="login" value="" size="15" placeholder="">
<div class="actions">
</div>
</div>
<div class="input-group">
<div class="label ">
<label for="password">Password</label>
</div>
<input id="password" class="password" type="password" name="password" value="" autocomplete="off" placeholder="">
<span class="password-eye-icon icon-eye hidden"></span>
<div class="actions">
<a href="/action/requestResetPassword" class="link show-request-reset-password">Forgot password?</a>
</div>
</div>
<div class="remember">
<div class="keepMeLogin">
<span class="label">Keep me logged in</span>
</div>
<div class="switch small-switch">
<input id="119162c4-9551-4055-99fe-48538e2570bc-remember" class="cmn-toggle cmn-toggle-round-flat" type="checkbox" name="remember" value="true">
<label class="tgl-btn" for="119162c4-9551-4055-99fe-48538e2570bc-remember"></label>
</div>
</div>
<div class="submit" disabled="disabled">
<input class="button submit primary" type="submit" name="loginSubmit" value="Login" disabled="disabled">
</div>
</form>POST /action/changePassword
<form action="/action/changePassword" method="post">
<div class="message error"></div>
<input type="hidden" name="submit" value="submit">
<div class="input-group">
<div class="label">
<label for="32ca9cb2-7bcf-451c-a718-7dacb5e529b9-old">Old Password</label>
</div>
<input id="32ca9cb2-7bcf-451c-a718-7dacb5e529b9-old" class="old" type="password" name="old" value="" autocomplete="off">
<span class="password-eye-icon icon-eye hidden"></span>
</div>
<div class="input-group">
<div class="label">
<label for="32ca9cb2-7bcf-451c-a718-7dacb5e529b9-new">New Password</label>
</div>
<input id="32ca9cb2-7bcf-451c-a718-7dacb5e529b9-new" class="pass-hint new" type="password" name="new" value="" autocomplete="off">
<span class="password-eye-icon icon-eye hidden"></span>
<div class="password-strength-indicator" data-min="8" data-max="20" data-strength="3">
<span class="text too-short">Too Short</span>
<span class="text weak">Weak</span>
<span class="text medium">Medium</span>
<span class="text strong">Strong</span>
<span class="text very-strong">Very Strong</span>
<span class="text too-long">Too Long</span>
</div>
</div>
<input class="button primary submit" type="submit" value="Submit" disabled="disabled">
</form>POST /action/registration
<form action="/action/registration" class="registration-form" method="post"><input type="hidden" name="redirectUri" value="/doi/10.1089/zeb.2019.1796">
<div class="input-group">
<div class="label">
<label for="4e824763-0b45-4b53-bcb3-79723a798efa.email">Email</label>
</div>
<input id="4e824763-0b45-4b53-bcb3-79723a798efa.email" class="email" type="email" name="email" value="">
</div>
<div class="submit">
<input class="button submit primary" type="submit" value="Register" disabled="disabled">
</div>
</form>POST /action/requestResetPassword
<form action="/action/requestResetPassword" class="request-reset-password-form" method="post"><input type="hidden" name="requestResetPassword" value="true">
<div class="input-group">
<div class="label">
<label for="d08ba088-273c-4e80-a5d6-e71647cef18b.email">Email</label>
</div>
<input id="d08ba088-273c-4e80-a5d6-e71647cef18b.email" class="email" type="text" name="email" value="" size="15">
</div>
<div class="password-recaptcha-ajax"></div>
<input class="button primary submit" type="submit" name="submit" value="Submit" disabled="disabled">
</form>POST /action/requestUsername
<form action="/action/requestUsername" method="post"><input type="hidden" name="requestUsername" value="requestUsername">
<div class="input-group">
<div class="label">
<label for="2b3925a2-1b57-45ab-a1cc-e6eb17123bf9.email">Email</label>
</div>
<input id="2b3925a2-1b57-45ab-a1cc-e6eb17123bf9.email" class="email" type="text" name="email" value="" size="15">
</div>
<div class="username-recaptcha-ajax"></div>
<input class="button primary submit" type="submit" name="submit" value="Submit" disabled="disabled">
<div class="center">
<a href="#" class="cancel">Close</a>
</div>
</form>Name: thisJournalQuickSearch — GET /action/doSearch
<form action="/action/doSearch" name="thisJournalQuickSearch" method="get">
<fieldset>
<legend class="sr-only">Quick Search in Journals</legend>
<div class="input-group option-0 option-journal"><label for="AllField374c2551-8302-4bf6-989c-6e4d27b968c20" class="sr-only">Enter words / phrases / DOI / ISBN / keywords / authors / etc</label>
<div class="autoComplete_wrapper" role="combobox" aria-owns="autoComplete_list_1" aria-haspopup="true" aria-expanded="false"><input type="search" autocomplete="off" id="AllField374c2551-8302-4bf6-989c-6e4d27b968c20" name="AllField"
placeholder="Enter words / phrases / DOI / ISBN / keywords / authors / etc" data-auto-complete-max-words="7" data-auto-complete-max-chars="32" data-contributors-conf="3" data-topics-conf="3" data-publication-titles-conf="3"
data-history-items-conf="3" value="" class="auto-complete" aria-controls="autoComplete_list_1" aria-autocomplete="both"></div><input type="hidden" name="SeriesKey" value="zeb">
</div>
<ul id="autoComplete_list_1" role="listbox" hidden="" class="autoComplete rlist"></ul><button type="submit" title="Search" class="btn quick-search__button icon-search"><span class="sr-only">Search</span><span>Search</span></button>
</fieldset>
</form>Name: defaultQuickSearch — GET /action/doSearch
<form action="/action/doSearch" name="defaultQuickSearch" method="get">
<fieldset>
<legend class="sr-only">Quick Search anywhere</legend>
<div class="input-group option-1 "><label for="AllField374c2551-8302-4bf6-989c-6e4d27b968c21" class="sr-only">Enter words / phrases / DOI / ISBN / keywords / authors / etc</label>
<div class="autoComplete_wrapper" role="combobox" aria-owns="autoComplete_list_2" aria-haspopup="true" aria-expanded="false"><input type="search" autocomplete="off" id="AllField374c2551-8302-4bf6-989c-6e4d27b968c21" name="AllField"
placeholder="Enter words / phrases / DOI / ISBN / keywords / authors / etc" data-auto-complete-max-words="7" data-auto-complete-max-chars="32" data-contributors-conf="3" data-topics-conf="3" data-publication-titles-conf="3"
data-history-items-conf="3" value="" class="auto-complete" aria-controls="autoComplete_list_2" aria-autocomplete="both"></div>
</div>
<ul id="autoComplete_list_2" role="listbox" hidden="" class="autoComplete rlist"></ul><button type="submit" title="Search" class="btn quick-search__button icon-search"><span class="sr-only">Search</span><span>Search</span></button>
</fieldset>
</form>Text Content
Back to Top
LOGIN TO YOUR ACCOUNT
Username
Password
Forgot password?
Keep me logged in
OpenAthens
New User
Institutional Login
CHANGE PASSWORD
Old Password
New Password
Too Short Weak Medium Strong Very Strong Too Long
PASSWORD CHANGED SUCCESSFULLY
Your password has been changed
CREATE A NEW ACCOUNT
Email
Returning user
Can't sign in? Forgot your password?
Enter your email address below and we will send you the reset instructions
Email
Cancel
If the address matches an existing account you will receive an email with
instructions to reset your password.
Close
REQUEST USERNAME
Can't sign in? Forgot your username?
Enter your email address below and we will send you your username
Email
Close
If the address matches an existing account you will receive an email with
instructions to retrieve your username
* This Journal
* This Journal
* Anywhere
Quick Search in Journals
Enter words / phrases / DOI / ISBN / keywords / authors / etc
SearchSearch
Quick Search anywhere
Enter words / phrases / DOI / ISBN / keywords / authors / etc
SearchSearch
Advanced Search
*
* Institutional Access
* 0
Home
* About Us
* Publications
Journal Collections
Biomedical ResearchBiotechnology and Regenerative MedicineMedicine and
SurgeryEnvironmental Research and PolicyIntegrative MedicineLaw and
PolicyNursingPublic Health Research and PolicySurgeryTechnology and
Engineering
Publications by Type
Journals (Print/Online)Journals (Open Access)Journals
(Video)Books/eBooksTrade MagazineseNewsletters
All Publications
A to ZRecommend a Title to Your Library
* For Authors
* Librarians
* Open Access
* Advertising
* Custom
ZebrafishVol. 17, No. 1
TechnoFish—MethodsFree Access
RAPID AND EFFICIENT LIVE ZEBRAFISH EMBRYO GENOTYPING
* Xue Zhang,
* Zhaojunjie Zhang,
* Qinshun Zhao, and
* Xin Lou
Xue Zhang
Model Animal Research Centre, Nanjing University, Nanjing, China.
*These authors contributed equally to this work.
Search for more papers by this author
,
Zhaojunjie Zhang
Model Animal Research Centre, Nanjing University, Nanjing, China.
*These authors contributed equally to this work.
Search for more papers by this author
,
Qinshun Zhao
Model Animal Research Centre, Nanjing University, Nanjing, China.
Search for more papers by this author
, and
Xin Lou
Address correspondence to: Xin Lou, PhD, Model Animal Research Centre, Nanjing
University, Room 403B, 12 Xuefu Road, Nanjing 210093, China
E-mail Address: xin.lou@nju.edu.cn
Model Animal Research Centre, Nanjing University, Nanjing, China.
Search for more papers by this author
Published Online:28 Jan 2020https://doi.org/10.1089/zeb.2019.1796
About
* Figures
* References
* Related
* Details
* Sections
* Abstract
* Ethics Approval
* Acknowledgments
* Authors' Contributions
* Disclosure Statement
* Funding Information
* Supplementary Material
* View article
* Supplemental Material
* PDF/EPUB
* View Supplemental Data
* Permissions & Citations
* Permissions
* Download Citations
* Track Citations
* Add to favorites
* Back To Publication
* Share
Share on
* Facebook
* Twitter
* Linked In
* Reddit
* Email
View article
* Supplemental Material
* PDF/EPUB
* View Supplemental Data
ABSTRACT
Most current methods for genotyping zebrafish embryos require sacrifice or
raising the animal to at least 1 month of age for fin amputation. These
limitations restrict the use of zebrafish and increase time and costs for
experiments. This article introduces an innovative method for genotyping live
zebrafish embryos. The method utilizes enzyme to extract a small amount of
genetic material from the skin tissue of the embryo. Then, using conventional
polymerase chain reaction (PCR) strategy, the embryo is genotyped. This approach
was successful >95% of the time while maintaining high viability (>90%) of the
embryo. This effective method can facilitate high-throughput screening and other
applications in zebrafish.
As a powerful vertebrate model system, zebrafish is being widely used in
research fields ranging from developmental biology, disease modeling, and drug
discovery. One of the advantages of zebrafish as a model is its amenability to
genetic manipulation, which has been dramatically improved with recently
developed genetic tools such as clustered regularly interspaced short
palindromic repeats (CRISPR) technology. However, using current methods for
genotyping zebrafish, embryos must be sacrificed; or fish must be raised to at
least 1 month old to have a fin clip collected.1 Methods have been described for
genotyping of live embryos; however, these require special instruments2,3 or
tedious surgical procedures.4,5 These limitations increase time and cost for
experiments and prohibit larger-scale phenotypic screens. Methods to enable fast
genotyping of live zebrafish embryos in a high-throughput manner would have
extensive applicability and practical significance.
As the zebrafish embryo has a remarkable capacity to regenerate, we set out to
establish a cost-efficient live embryo genotyping method based on limited
proteinase digestion. First, we tested the DNA extraction ability of different
types of proteinases on 72 h postfertilization (hpf) embryos in embryo culture
medium. To enhance the digestion efficacy, the samples were incubated at 37°C
and constantly mixed on a multiwell plate shaker (Fig. 1A). The results showed
both pronase and proteinase K could release DNA from embryo skin tissue, with
embryos treated by proteinase K showing higher viability. We, therefore,
optimized the procedure with proteinase K (Supplementary Fig. S1A). To further
improve the genotyping sensitivity, we prepared four different types of buffer
after literature review and compared the DNA extraction efficiency and embryo
viability for each. The results showed that when using 30 mM Tris (pH8.0),
genotyping polymerase chain reaction (PCR) could be successfully performed in
90% of samples, with a 90% survival rate of genotyped embryos (Fig. 1B and
Supplementary Fig. S1B). For applications, including drug screens, mutant
genotyping, and genetic screens, researchers require genotype information as
rapidly as possible. To minimize the damage of enzyme treatment to the early
stage embryo, we optimized the concentration of proteinase K, treatment time,
and shaking speed for embryos from 24 to 96 hpf (Supplementary Fig. S1C–F) and
listed the recommended treatment conditions in a detailed protocol
(Supplementary Data). To test the quality of DNA obtained from the method, we
deployed PCR for a variety of amplicon and DNA sequences, and found that up to
1.2 kb could be successfully amplified (Fig. 1C and Supplementary Table S1). We
also assessed the possible adverse effects of the treatment on larva behavior,
fish survival, and fecundity (Supplementary Fig. S2), with no significant
effects observed.
FIG. 1. Rapid and efficient live zebrafish embryo genotyping. (A) The step
procedure for the method. (B) Representative image of genotyped zebrafish larva
at 7 dpf. Lateral view, head to left. (C) Representative PCR results. dpf, days
postfertilization; NC, negative control; PC, positive control; PCR, polymerase
chain reaction.
To further enhance the throughput of this method, we established a two-round
procedure. At 48 hpf, up to eight embryos could be pooled into 1 well of a
24-well plate, and the mixed DNA was prepared and tested in first-round
genotyping. The embryos from positive pools were cultured to allow recovery. At
96 hpf, these embryos were individually re-genotyped (Supplementary Fig. S3).
This procedure (see note in the protocol) is suitable for genotyping screens
especially those with a low germline transmission rate (such as
CRISPR/Cas9-mediated knock-in, base-editing, and large fragment deletion), such
that up to 800 embryos could be processed by one person in one round of
genotyping.
Our results show that this method provides a fast, cost-efficient, and
high-throughput compatible solution to genotyping live zebrafish embryos. The
genetic material obtained from this method is suitable for detecting SNPs,
transgenes, and mutations, including single base-pair changes, insertions, or
deletions. To our knowledge, this is the first report of a limited digestion
method and could facilitate the use of zebrafish embryos in biomedical research.
ETHICS APPROVAL
Zebrafish were housed and handled as per Nanjing University Laboratory Animal
Services guidelines. All the protocols involving the use of animals were in
accordance with approved guidelines of the Institutional Animal Care and Use
Committee of the Nanjing University.
ACKNOWLEDGMENTS
We thank Prof. Ian C. Scott at University of Toronto, for help in editing the
article, and members of Di Chen Laboratory at Nanjing University, for the help
during this project.
AUTHORS' CONTRIBUTIONS
X.L. and Q.Z. conceived and supervised the project. X.Z. and Z.Z. performed
experiments and analyzed the data. X.L. wrote the article.
DISCLOSURE STATEMENT
No competing financial interests exist.
FUNDING INFORMATION
This research was undertaken, in part, thanks to grant funding from National
Natural Science Foundation of China (NSFC 31671505 and 31970765 to X.L).
SUPPLEMENTARY MATERIAL
Supplementary Data
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Table S1
* Figures
* References
* Related
* Details
REFERENCES
* 1. Parant JM, George SA, Pryor R, Wittwer CT, Yost HJ. A rapid and efficient
method of genotyping zebrafish mutants. Dev Dyn 2009;238:3168–3174. Crossref,
Medline, Google Scholar
* 2. Samuel R, Stephenson R, Roy P, Pryor R, Zhou L, Bonkowsky JL, et al.
Microfluidic-aided genotyping of zebrafish in the first 48 h with 100%
viability. Biomed Microdevices 2015;17:43. Crossref, Medline, Google Scholar
* 3. Lambert CJ, Freshner BC, Chung A, Stevenson TJ, Bowles DM, Samuel R, et
al. An automated system for rapid cellular extraction from live zebrafish
embryos and larvae: development and application to genotyping. PLoS One
2018;13:e0193180. Crossref, Medline, Google Scholar
* 4. Xing L, Quist TS, Stevenson TJ, Dahlem TJ, Bonkowsky JL. Rapid and
efficient zebrafish genotyping using PCR with high-resolution melt analysis.
J Vis Exp 2014;e51138. Medline, Google Scholar
* 5. Kosuta C, Daniel K, Johnstone DL, Mongeon K, Ban K, Leblanc S, et al..
High-throughput DNA extraction and genotyping of 3dpf zebrafish larvae by fin
clipping. J Vis Exp 2018. DOI: 10.3791/58024. Crossref, Medline, Google
Scholar
* Cited by
* Improved selection of zebrafish CRISPR editing by early next-generation
sequencing based genotyping
27 January 2023 | Scientific Reports, Vol. 13, No. 1
* Integration of cooperative and opposing molecular programs drives
learning-associated behavioral plasticity
27 March 2023 | PLOS Genetics, Vol. 19, No. 3
* blf and the drl cluster synergistically regulate cell fate commitment
during zebrafish primitive hematopoiesis
14 December 2022 | Development, Vol. 149, No. 24
* A minimally invasive fin scratching protocol for fast genotyping and early
selection of zebrafish embryos
30 December 2022 | Scientific Reports, Vol. 12, No. 1
* Genetic therapy in a mitochondrial disease model suggests a critical role
for liver dysfunction in mortality
21 November 2022 | eLife, Vol. 11
* DPP9 deficiency: An inflammasomopathy that can be rescued by lowering
NLRP1/IL-1 signaling
Science Immunology, Vol. 7, No. 75
* The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA
Programming of Human Cells In Vitro and Zebrafish Disease Models In Vivo
* Ankit Sabharwal,
* Bibekananda Kar,
* Santiago Restrepo-Castillo,
* Shannon R. Holmberg,
* Neal D. Mathew,
* Benjamin Luke Kendall,
* Ryan P. Cotter,
* Zachary WareJoncas,
* Christoph Seiler,
* Eiko Nakamaru-Ogiso,
* Karl J. Clark, and
* Stephen C. Ekker
16 December 2021 | The CRISPR Journal, Vol. 4, No. 6
* A deterministic genotyping workflow reduces waste of transgenic
individuals by two-thirds
28 July 2021 | Scientific Reports, Vol. 11, No. 1
* Modeling Lysosomal Storage Diseases in the Zebrafish
6 May 2020 | Frontiers in Molecular Biosciences, Vol. 7
* Recommended
* ADDITIVE MANUFACTURING CAN ASSIST IN THE FIGHT AGAINST COVID-19 AND OTHER
PANDEMICS AND IMPACT ON THE GLOBAL SUPPLY CHAIN
* Eneko Larrañeta,
* Juan Dominguez-Robles, and
* Dimitrios A. Lamprou
Vol. 7, No. 3 June 2020
* UROLOGIC SURGERY AND COVID-19: HOW THE PANDEMIC IS CHANGING THE WAY WE
OPERATE
* James E. Steward,
* Weston R. Kitley,
* C. Max Schmidt, and
* Chandru P. Sundaram
Vol. 34, No. 5 May 2020
* ROLE OF TISSUE ENGINEERING IN COVID-19 AND FUTURE VIRAL OUTBREAKS
* Alexander M. Tatara
Vol. 26, No. 9-10 May 2020
* SOFT ROBOTICS IN MINIMALLY INVASIVE SURGERY
* Mark Runciman,
* Ara Darzi, and
* George P. Mylonas
Vol. 6, No. 4 August 2019
* COVID-19: GENE TRANSFER TO THE RESCUE?
* Luk H. Vandenberghe
Vol. 31, No. 11-12 June 2020
Volume 17Issue 1
Feb 2020
Information
Copyright 2020, Mary Ann Liebert, Inc., publishers
--------------------------------------------------------------------------------
To cite this article:
Xue Zhang, Zhaojunjie Zhang, Qinshun Zhao, and Xin Lou.Rapid and Efficient Live
Zebrafish Embryo Genotyping.Zebrafish.Feb
2020.56-58.http://doi.org/10.1089/zeb.2019.1796
* Published in Volume: 17 Issue 1: January 28, 2020
* Online Ahead of Print:December 17, 2019
Keywords
* basic protocol
* genotyping
* zebrafish
* mutation
* transgenes
--------------------------------------------------------------------------------
PDF download
--------------------------------------------------------------------------------
back
Close Figure Viewer
Browse All FiguresReturn to FigureChange zoom levelZoom inZoom out
Previous FigureNext Figure
Caption
Back to Top
*
*
*
PUBLICATIONS
* Publications A-Z
* Journal Collections
* Publications by Type
* Recommend a Title
FOR AUTHORS
* Fees and Options
* Publishing Open Access
* Submission Guidelines
* Policies
LIBRARIANS
* Our Journals
* Account Support
* Archive
* Terms & Conditions
* Resources
* Liebert Link Newsletter
* Contact
OPEN ACCESS
* Open Option
* Open Access Journals
* Publishing Services
* FAQs
* Contact
CORPORATE CAPABILITIES
* Custom Publications
* Interactive Media
* Other Opportunities
* Reprints & ePrints
ADVERTISING
COMPANY
CUSTOMER SUPPORT
CONTACT US
PRIVACY POLICY
© 2023 Mary Ann Liebert, Inc., publishers. All rights reserved, USA and
worldwide.
Call us toll free at (800) M-LIEBERT (800-654-3237).
⊠
We use cookies to give you a better experience on liebertpub.com. By continuing
to use our site, you are agreeing to the use of cookies as set in our Cookie
Policy.OK
✓
Thanks for sharing!
AddToAny
More…
Close crossmark popup