This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

<ol>
	<li>Yergeau, D., Kelley, C. ., Zhu, H., Kuliyev, E., Mead, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+Genetic+Screens+in+Xenopus.+Using+Transposon-Mediated+Insertional+Mutagenesis.">Forward Genetic Screens in Xenopus. Using Transposon-Mediated Insertional Mutagenesis.</a> Methods in Molecular Biology. 917, 111-127 (2012).</li><li>Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.+as+a+Model+Organism+for+Genetics+and+Genomics:+Past,+Present+and+Future.">Xenopus tropicalis. as a Model Organism for Genetics and Genomics: Past, Present and Future.</a> Methods in Molecular Biology. 917, 3-15 (2012).</li><li>Nakayama, T., Fish, M., Fisher, M., Oomen-Hajagos, J., Thomsen, G., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+Efficient+CRISPR/Cas9-Mediated+Targeted+Mutagenesis+in+Xenopus+tropicalis.">Simple and Efficient CRISPR/Cas9-Mediated Targeted Mutagenesis in Xenopus tropicalis.</a> Genesis. 51, 835-843 (2013).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis.">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.</a> Biology Open. 1 (12), 1273-1276 (2012).</li><li>Zhao, Y., Ishibashi, S., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetic+Studies+Using+Antisense+Morpholino+Oligonucleotides.">Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides.</a> Methods in Molecular Biology. 917, 143-154 (2012).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injected+Xwnt-8.+RNA+acts+early+in+Xenopus.+embryos+to+promote+formation+of+a+vegetal+dorsalizing+center.">Injected Xwnt-8. RNA acts early in Xenopus. embryos to promote formation of a vegetal dorsalizing center.</a> Cell. 67, 753-765 (1991).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin.,+a+new+dorsalizing+factor+localized+in+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin., a new dorsalizing factor localized in the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Plautz, C., Zirkle, B., Deshote, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+induction+of+anterior+head+ectodermal+properties+in+Xenopus.+embryos+are+mediated+by+transcriptional+cofactor+ldb1.">Early stages of induction of anterior head ectodermal properties in Xenopus. embryos are mediated by transcriptional cofactor ldb1.</a> Developmental Dynamics. 243 (12), 1606-1618 (2014).</li><li>Servetnick, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+neural+and+lens+competence+in+Xenopus.+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus. ectoderm: evidence for an autonomous developmental timer.</a> Development. 112, 177-188 (1991).</li><li>Nieuwkoop, P., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li><li>Lemaire, P., Garrett, N., Gurdon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+Siamois.,+a+Xenopus.+homeobox+gene+expressed+in+dorsal-vegetal+cells+of+blastulae+and+able+to+induce+a+complete+secondary+axis.">Expression cloning of Siamois., a Xenopus. homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.</a> Cell. 81, 85-94 (1995).</li><li>Lustig, K., Kroll, K., Sun, E., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+a+Xenopus+T.-related+gene+(Xombi.)+involved+in+mesodermal+patterning+and+blastopore+lip+formation.">Expression cloning of a Xenopus T.-related gene (Xombi.) involved in mesodermal patterning and blastopore lip formation.</a> Development. 122, 4001-4012 (1996).</li><li>Hsu, D., Economides, A., Wang, X., Eimon, P., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus.+dorsalizing+factor+Gremlin+identifies+a+novel+family+of+secreted+proteins+that+antagonize+BMP+activities.">The Xenopus. dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.</a> Molecular Cell. 1 (5), 673-683 (1998).</li><li>Lustig, K., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+oocyte+expression+assay+to+reconstitute+inductive+signaling.">Use of an oocyte expression assay to reconstitute inductive signaling.</a> Proc. Natl. Acad. Sci. USA. 92, 6234-6238 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE Science Education Database. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests.">Basic Methods in Cellular and Molecular Biology. 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(39), e1932 (2010).</li><li>Sive, H., Grainger, R., Harland, 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> Early development of.Xenopus laevis.: A laboratory manual. , (2000).</li><li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activation+during+early+stages+of+lens+induction+in+Xenopus.">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li><li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxe+view+of+lens+development+and+disease.">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li><li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism.">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li><li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project.">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li><li>Karpinka, 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=Xenbase,+the+Xenopus.+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li><li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis.">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469 (2013).</li></ol>

The authors declare that they have no competing financial interests.

השיטה המתוארת כאן לשיבוט הפונקציונלי של גנים מסוגלים גרימת תגובה בהאאקטודרם מוסמך יכולה לשמש כדי לזהות מגוון רחב של מוצרי גן. שיטה זו מרחיבה על העבודה האחרונה על ידי שילוב של מבחני וישכנע רקמה עם טכניקות שיבוט ביטוי. אנו מנצלים את המסלולים המטבוליים של ביצית Xenopus</e...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table><tbody><tr><td>12/101 Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>12/101</td><td>Monoclonal antibody for detection of muscle tissue</td></tr><tr><td>20x SSC Buffer</td><td>Sigma</td><td>S6639</td><td>for ISH</td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td>for ISH</td></tr><tr><td>Anti-Dig-AP</td><td>Roche</td><td>11093274910</td><td>for ISH</td></tr><tr><td>Aurum Plasmid Mini Kit</td><td>Bio-Rad</td><td>732-6400</td><td>Plasmid DNA purification</td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td>for ISH</td></tr><tr><td>BM Purple</td><td>Roche</td><td>11442074001</td><td>for ISH</td></tr><tr><td>Boekel Hybridization Oven</td><td>Fisher Scientific</td><td>13-245-121</td><td>for ISH</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma</td><td>HT10132</td><td>for ISH</td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td>for OCM</td></tr><tr><td>CHAPS</td><td>Sigma</td><td>C3023</td><td>for ISH</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>Defolliculation of oocytes</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C121800</td><td>Dejelly embryos</td></tr><tr><td>DEPC-H2O</td><td>Fisher Scientific</td><td>BP5611</td><td>for ISH</td></tr><tr><td>Dig-RNA Labeling Mix</td><td>Roche</td><td>11277073910</td><td>for ISH probes</td></tr><tr><td>Dumont #5 forceps</td><td>World Precision Instruments</td><td>500233</td><td>for Vitelline envelope removal</td></tr><tr><td>Ethyl 3-aminobenzoate</td><td>Sigma</td><td>A5040</td><td>MS222 anesthetic</td></tr><tr><td>Ficoll PM 400</td><td>Sigma</td><td>F4375</td><td>for Injection media</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td>for ISH</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma</td><td>G1914</td><td>for OCM</td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>4878</td><td>3.5&quot; long, I,D, 0.530 mm</td></tr><tr><td>Glass sample vials</td><td>Fisher Scientific</td><td>06-408B</td><td>for ISH</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Hair affixed in pasteur pipette for tissue manipulation</td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td>for ISH</td></tr><tr><td>Injector Nanoliter 2010</td><td>World Precision Instruments</td><td>Nanoliter 2010</td><td>Microprocessor-controlled microinjector</td></tr><tr><td>Instant Ocean</td><td>Carolina</td><td>972433</td><td>Aquarium Salt for frog recovery</td></tr><tr><td>IRBG XGC Xenopus verified full-length cam cDNA</td><td>Source Bioscience</td><td>989_IRBG</td><td>cDNA library&amp;nbsp;</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/ml Ampicillin</td><td>Teknova</td><td>L5004</td><td>150 mm pre-poured LB-Amp plates for sib selection</td></tr><tr><td>LB Luria Broth</td><td>Teknova</td><td>L8650</td><td>LB for collecting colonies in sib selection from plates and dilution of cultures</td></tr><tr><td>Magnetic mRNA Isolation Kit</td><td>New England BioLabs</td><td>S1550S</td><td>for isolation of poly(A)-enriched RNA</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375</td><td>for ISH</td></tr><tr><td>Manual Microfil Micromanipulator</td><td>World Precision Instruments</td><td>M3310R</td><td>Manual micromanipulator</td></tr><tr><td>Nutating Mixer</td><td>Fisher Scientific</td><td>22-363-152</td><td>Rocker for ISH</td></tr><tr><td>Permoplast</td><td>Nasco</td><td>SB33495M</td><td>Clay for injection and dissection dishes</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>P5368</td><td>for ISH</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877</td><td>to stimulate oocyte development</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>PVP40</td><td>for ISH</td></tr><tr><td>Programmable Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Micropipette needle puller</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td>for ISH</td></tr><tr><td>pTnT Vector</td><td>Promega</td><td>L5610</td><td>cDNA library construction</td></tr><tr><td>Riboprobe Combination System</td><td>Promega</td><td>P1450</td><td>in vitro transcription</td></tr><tr><td>Superscript Full Length cDNA Library Construction Kit</td><td>Life Technologies</td><td>18248013</td><td>kit for cDNA library construction</td></tr><tr><td>Sutures, 3-0 silk</td><td>Fisher Scientific</td><td>19-037-516</td><td>Suture thread and needle for post-oocyte removal</td></tr><tr><td>Torula RNA</td><td>Sigma</td><td>R3629</td><td>for ISH</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>T1502</td><td>for ISH</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td>for ISH</td></tr><tr><td>Universal RiboClone cDNA Synthesis System</td><td>Promega</td><td>C4360</td><td>alternative kit for cDNA library construction</td></tr><tr><td>Xenopus Full ORF Entry Clones - ORFeome Collaboration</td><td>Source Bioscience</td><td>5055_XenORFeome</td><td>ORFeome Clones</td></tr><tr><td>XL2-Blue Ultracompetent Cells</td><td>Agilent Technologies</td><td>200150</td><td>cells for transformation of cDNA library</td></tr></tbody></table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

בתגובה לביטוי של mRNA מוזרק לתוך ביציות, להגיב רקמת כובע חיה assayed לביטוי של otx2 ידי הכלאה באתר (איור 2 ולוח 1); otx2 בא לידי ביטוי בהאאקטודרם העדשה המשוערת (PLE) מסגירת צינור עצבית באמצעות placode העדשה עיבוי 19. עם זאת, מאז otx2 בא ל...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

שיבוט פונקציונלי שימוש<em&gt; Xenopus</emמערכת ביטוי ביצית&gt;

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

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Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K Views.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Video: Functional Cloning Using a Xenopus Oocyte Expression System

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

ומניפולציה<em&gt; במבחנה</em&gt; ההבשלה של<em&gt; Laevis Xenopus</em&gt; ביציות, ואחריו הזרקת Intracytoplasmic זרע, לחקר התפתחות העוברית

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

באמצעות ניתוח של Confocal<em&gt; Laevis Xenopus</em&gt; לחקירת מאפננים של עירובים מורפוגן Wnt וששש

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

השתלת תא נבט הקדמוני עבור מבוססת על CRISPR/Cas9 וקפיצת המדרגה של צפרדע רפואית

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Genetics

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

כמו תא גזע<em&gt; Xenopus</em&gt; Explants העובריים לחקר תכונות המוקדמות עצבי התפתחותית<em&gt; במבחנה</em&gt; ו<em&gt; In vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection של ביציות Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biology

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

הזרקת דנ א לתוך eyebuds ב xenopus זריזה העוברים והדמיה של gfp המבטא האופטי axonal הביע בשלמותו, חי xenopus tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

הפריה של Xenopus ביציות באמצעות שיטת העברה מארח

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-תא הדמיה וניתוח כמותי של תאים אפיתל עובריים ב Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

הפקה של טראנסגנטי Xenopus laevis על ידי שילוב אנזימים מתווכת הגבלת והשתלות גרעיני

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

יישום של עכבר פרתנוגנטי תאי גזע עובריים הפלואיד כתחליף של זרע

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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