Name: primordial germ cell transplantation for crispr/cas9-based leapfrogging in xenopus
Uploaded: 2018-02-01T13:30:00
Description: Watch this Scientific Journal Video about Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus at JoVE.com

This work was performed with the support of a grant, 5R21HD080684-02, from the National Institute of Child Health and Human Development. The author wishes to thank Ken Cho for his continuing enthusiasm and support. The author would also like to acknowledge Bruce Blumberg for use of his camera, Rebekah Charney, for the critical reading of the manuscript, and Sean McNamara and Marcin Wlizla at the National Xenopus Resource (RRID:SCR_013731), for the valuable conversations regarding X. tropicalis feeding and animal care regimens.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.
1. Preparations for PGC Transplantation
<ol>
	<li>Prepare dissection tools in advance, as previously described23. 
	NOTE: Eyebrow hair knives are used to make incisions with the assistance of a hair loop to stabilize the embryo while performing the surgeries. Eyebrow hairs and hair loops are glued into borosilicate glass Pasteu...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+evolution+in+the+allotetraploid+frog+Xenopus+laevis.">Genome evolution in the allotetraploid frog Xenopus laevis.</a> Nature. 538 (7625), 336-343 (2016).</li><li>Blitz, I. L., Biesinger, J., Xie, X., Cho, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biallelic+genome+modification+in+F(0)+Xenopus+tropicalis+embryos+using+the+CRISPR/Cas+system.">Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system.</a> Genesis. 51 (12), 827-834 (2013).</li><li>Nakayama, T., Fish, M. B., Fisher, M., Oomen-Hajagos, J., Thomsen, G. H., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 (12), 835-843 (2013).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li><li>Wang, F., Shi, Z., Cui, Y., Guo, X., Shi, Y. B., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+in+Xenopus+laevis+using+CRISPR/Cas9.">Targeted gene disruption in Xenopus laevis using CRISPR/Cas9.</a> Cell Biosci. 5, 15 (2015).</li><li>Jaffe, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c21orf59/kurly+Controls+Both+Cilia+Motility+and+Polarization.">c21orf59/kurly Controls Both Cilia Motility and Polarization.</a> Cell Rep. 14 (8), 1841-1849 (2016).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+editing+of+genes+involved+in+neural+crest+development+using+the+CRISPR/Cas9+system+in+Xenopus+embryos.">Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.</a> Cell Biosci. 6, 22 (2016).</li><li>Naert, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+mediated+knockout+of+rb1+and+rbl1+leads+to+rapid+and+penetrant+retinoblastoma+development+in+Xenopus+tropicalis.">CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis.</a> Sci Rep. 6 (35264), (2016).</li><li>Ratzan, W., Falco, R., Salanga, C., Salanga, M., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+Xenopus+laevis+F1+albino+J+strain+by+genome+editing+and+oocyte+host-transfer.">Generation of a Xenopus laevis F1 albino J strain by genome editing and oocyte host-transfer.</a> Dev Biol. 426 (2), (2016).</li><li>Bhattacharya, D., Marfo, C. A., Li, D., Lane, M., Khokha, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9:+An+inexpensive,+efficient+loss+of+function+tool+to+screen+human+disease+genes+in+Xenopus.">CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.</a> Dev Biol. 408 (2), 196-204 (2015).</li><li>Shigeta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+analysis+of+gene+function+using+CRISPR-Cas9+in+Xenopus+tropicalis+founders.">Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.</a> Genes Cells. 21 (7), 755-771 (2016).</li><li>Hontelez, S., 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=Embryonic+transcription+is+controlled+by+maternally+defined+chromatin+state.">Embryonic transcription is controlled by maternally defined chromatin state.</a> Nat Commun. 6, 10148 (2015).</li><li>Peshkin, L., 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=On+the+Relationship+of+Protein+and+mRNA+Dynamics+in+Vertebrate+Embryonic+Development.">On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development.</a> Dev Cell. 35 (3), 383-394 (2015).</li><li>Owens, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Absolute+RNA+Copy+Numbers+at+High+Temporal+Resolution+Reveals+Transcriptome+Kinetics+in+Development.">Measuring Absolute RNA Copy Numbers at High Temporal Resolution Reveals Transcriptome Kinetics in Development.</a> Cell Rep. 14 (3), 632-647 (2016).</li><li>Blitz, I. L., Fish, M. B., Cho, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leapfrogging:+primordial+germ+cell+transplantation+permits+recovery+of+CRISPR/Cas9-induced+mutations+in+essential+genes.">Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.</a> Development. 143 (15), 2868-2875 (2016).</li><li>Blackler, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+germ-cells+in+Xenopus+laevis.">Transfer of germ-cells in Xenopus laevis.</a> Nature. 185, 859-860 (1960).</li><li>Blackler, A. W., Fischberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+primordial+germ-cells+in+Xenopus+laevis.">Transfer of primordial germ-cells in Xenopus laevis.</a> J Embryol. Exp Morph. 9, 634-641 (1961).</li><li>Bounoure, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> L'origine des cellules reproductrices et le problem de la lignee germinale. , (1939).</li><li>Nieuwkoop, P. D., Sutasurya, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primordial Germ Cells in the Chordates: Embryogenesis and phylogenesis. , (1979).</li><li>Heasman, J., Holwill, S., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods Cell Biol. 36, 213-230 (1991).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+dissection+and+micromanipulation+tools.">Embryo dissection and micromanipulation tools.</a> CSH Protoc. , (2007).</li><li>Nakayama, T., 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=Cas9-based+genome+editing+in+Xenopus+tropicalis.">Cas9-based genome editing in Xenopus tropicalis.</a> Methods Enzymol. 546, 355-375 (2014).</li><li>Ogino, H., McConnell, W. B., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+transgenesis+in+Xenopus+using+I-SceI+meganuclease.">High-throughput transgenesis in Xenopus using I-SceI meganuclease.</a> Nat Protoc. 1 (4), 1703-1710 (2006).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis.: a laboratory manual. , (2000).</li><li>Kay, B. K., Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+oocytes+and+embryos.">Injection of oocytes and embryos.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology (Methods in Cell Biology Vol. 36). , (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): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metomorphosis. , (1994).</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Res. 42 (22), e168 (2014).</li><li>Sander, V., Reversade, B., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+opposing+homeobox+genes+Goosecoid+and+Vent1/2+self-regulate+Xenopus+patterning.">The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning.</a> EMBO J. 26 (12), 2955-2965 (2007).</li><li>Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+using+a+high-efficiency+TALEN+system.">In vivo genome editing using a high-efficiency TALEN system.</a> Nature. 491 (7422), 114-118 (2012).</li><li>Kim, J. M., Kim, D., Kim, S., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+with+CRISPR-Cas-derived+RNA-guided+endonucleases.">Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.</a> Nat Commun. 5, 3157 (2014).</li><li>Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+human+cells+with+zinc+finger+nucleases+constructed+via+modular+assembly.">Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.</a> Genome Res. 19 (7), 1279-1288 (2009).</li><li>Qiu, P., Shandilya, H., D'Alessio, J. M., O'Connor, K., Durocher, J., Gerard, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+detection+using+Surveyor+nuclease.">Mutation detection using Surveyor nuclease.</a> Biotechniques. 36 (4), 702-707 (2004).</li><li>Ota, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+identification+of+TALEN-mediated+genome+modifications+using+heteroduplex+mobility+assays.">Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.</a> Genes Cells. 18 (6), 450-458 (2013).</li><li>Dahlem, T. 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=Simple+Methods+for+Generating+and+Detecting+Locus-+Specific+Mutations+Induced+with+TALENs+in+the+Zebrafish+Genome.">Simple Methods for Generating and Detecting Locus- Specific Mutations Induced with TALENs in the Zebrafish Genome.</a> PLoS Genet. 8 (8), e1002861 (2012).</li><li>Ramlee, M. K., Yan, T., Cheung, A. M., Chuah, C. T., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genotyping+of+CRISPR/Cas9-mediated+mutants+using+fluorescent+PCR-capillary+gel+electrophoresis.">High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.</a> Sci Rep. 5, 15587 (2015).</li><li>Bhattacharya, D., Marfo, C. A., Li, D., Lane, M., Khokha, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9:+An+inexpensive,+efficient+loss+of+function+tool+to+screen+human+disease+genes+in+Xenopus.">CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.</a> Dev Biol. 408 (2), 196-204 (2015).</li><li>Yu, C., Zhang, Y., Yao, S., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+Based+Protocol+for+Detecting+Indel+Mutations+Induced+by+TALENs+and+CRISPR/Cas9+in+Zebrafish.">A PCR Based Protocol for Detecting Indel Mutations Induced by TALENs and CRISPR/Cas9 in Zebrafish.</a> PLoS ONE. 9 (6), e98282 (2014).</li><li>Mock, U., Hauber, I., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+to+assess+gene-editing+frequencies+(GEF-dPCR)+mediated+by+designer+nucleases.">Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.</a> Nat Protoc. 11 (3), 598-615 (2016).</li><li>Varshney, G. K., 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=High-throughput+gene+targeting+and+phenotyping+in+zebrafish+using+CRISPR/Cas9.">High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.</a> Genome Res. 25 (7), 1030-1042 (2015).</li><li>Shi, J., Wang, E., Milazzo, J. P., Wang, Z., Kinney, J. B., Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+cancer+drug+targets+by+CRISPR-Cas9+screening+of+protein+domains.">Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.</a> Nat Biotechnol. 33 (6), 661-667 (2015).</li><li>Koebernick, K., Loeber, J., Arthur, P. 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This report provides a detailed protocol for the transplantation of vegetal tissue containing PGCs. Transplantation of PGCs is used in conjunction with genome-editing technologies (e.g., CRISPR/Cas9) to modify the germline of an animal while maintaining nearly all of its somatic tissues as genetically wild type. For leapfrogging to be successful, there are a number of critical factors to consider prior to performing performing transplantations.
To ensure the complete replacement of th...

How genotype encodes phenotype has been a major question in biology since the rediscovery of Mendel&#39;s laws. An understanding of the roles of genes, their regulation and interactions within gene networks, and the functions of encoded products promises to provide tools for uncovering new biology and ameliorating disease states. For over half a century1, the African clawed frog, Xenopus, has been a leading model for studies on a wide variety of topics in basic biology and biomedicine, including the genetic control of development. Historically, most research on Xenopus has used the allotetraploid frog, X. laevis, but more recently, due to its diploidy, X. tropicalis has been developed as an amphibian genetic model. Complete genome sequences have been assembled from both Xenopus species2,3. The &#34;frog community&#34; is now at a turning point where basic genome modification technology permits the study of gene function, virtually at will. Programmable CRISPR/Cas9 endonucleases have made the mutagenesis of genes highly efficient, with biallelic mutation possible in most cells of the animal4,5,6,7,8,9,10,11. These studies, underscored by Bhattacharya et al.12 and Shigeta et al.13, have shown that the function of many genes can be studied by mutagenesis in F0 mosaic animals. This approach has many advantages; however, Cas9-sgRNA-microinjected embryos often display variable phenotypes due to incomplete loss of function (LOF). More significantly, the generation of mutant lines is highly advantageous for some applications&#8212;in particular when studying genes that have a maternal mRNA contribution. Maternal mRNAs and proteins, and their influences on epigenetics, persist for an extended period into embryogenesis14,15,16, rendering the early developmental contributions of many genes refractory to F0 analyses. Therefore, other LOF approaches are required.
When seeking to create mutant lines, the path to obtaining homozygous LOF embryos has several obstacles. First, efficient mutagenesis to produce biallelic mutations can be disadvantageous because loss of essential gene functions results in failure to survive to sexual maturity, interfering with the production of a viable line. A common solution is the careful titration of the amount of Cas9-sgRNA delivered. Here, the goal is to achieve a balance between reducing lethality while also maximizing germline mutagenesis efficiency. A second problem arises from the standard breeding scheme, where phenotypic analyses are deferred until the F2 generation. Using the standard approach, sexually mature F0 animals that transmit mutant alleles through the germline are outcrossed to produce F1 heterozygous &#34;carriers&#34;, which are then grown to sexual maturity. Two F1 heterozygotes are then intercrossed to produce F2 mutant embryos at an expected Mendelian frequency of 25%. Thus, two generations of breeding are necessary for analysis of mutant phenotypes. Mutant animals could be either homozygotes or compound heterozygotes (i.e., progeny containing two different mutant alleles, which depends on the genotypes of the parental animals used in the F1 intercross).
These obstacles can be overcome by confining programmable nuclease-mediated mutagenesis to germ cells, which underlies a new method called leapfrogging17. Leapfrogging has two main components: (1) the microinjection of Cas mRNA together with sgRNA, or nuclease-sgRNA complexes (or, in principle, TALENs or zinc finger nucleases) at the single-cell stage to efficiently mutagenize embryonic genomes, followed by (2) the transplantation of PGCs into wildtype sibling embryos, where the endogenous PGCs were removed. When both steps are efficient, complete germline replacement with mutant germ cells can be obtained. Blackler demonstrated in the early 1960s that Xenopus PGCs could be transplanted between embryos at the late neurula and early tailbud stages18,19. For leapfrogging, Blackler&#39;s approach was modified by performing the transplantations at the blastula stage17, when PGCs are localized in the vegetal pole of the embryo20,21. Transplantation before gastrulation has two main advantages. First, the engraftment of transplants and the subsequent normal development was found to be more efficient when the transplantation is performed at the blastula stage (unpublished observations). Second, by performing blastula-stage transplantations shortly after zygotic transcription has begun, one can avoid the lethality arising from developmental gene mutations that disrupt gastrulation or that otherwise lead to malformed late neurulae. PGC transplant-bearing (&#34;leapfrogged&#34;) embryos are grown to sexual maturity, and intercrosses between these F0 animals have demonstrated that, in many cases, 100% of the F1 progeny display the LOF phenotype (most being compound heterozygotes), indicating complete germline replacement with the targeted mutations.
It is expected that leapfrogging will accelerate genetic approaches in Xenopus. Leapfrogging also provides an alternative to the &#34;host transfer&#34; method22 for the LOF analysis of maternal-effect genes (unpublished observations). In the current publication, a detailed description of the method, especially focusing on PGC transplantation, is presented in X. tropicalis (with minor modifications for X. laevis). The transplantation of PGCs is demonstrated here to facilitate a more rapid transfer of this technology to other laboratories working with Xenopus. The principles of this method should be successful in other amphibians (e.g., urodeles), and organism-specific modifications in the methodology should allow for application to many other animals in which efficient mutagenesis can be accomplished and PGCs are readily transplantable.

<table>
	<tbody>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20 or 11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyebrow hair knife</td>
			<td></td>
			<td></td>
			<td>Homemade</td>
		</tr>
		<tr>
			<td>Hair loop</td>
			<td></td>
			<td></td>
			<td>Homemade</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, borosilicate glass</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Krazy Glue (Cyanoacrylate-based)</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td>KG581</td>
			<td>To affix eyebrow hair and hair loops into Pasteur pipettes, other similar glues can be used.</td>
		</tr>
		<tr>
			<td>Oligodeoxynucleotides, custom ordered</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom ordered</td>
			<td>Template oligos for sgRNA synthesis, see Nakayama et al., 2014 for design details.</td>
		</tr>
		<tr>
			<td>Megascript T7 kit</td>
			<td>Ambion/ThermoFisher</td>
			<td>AM1334</td>
			<td>Or use Megashortscript T7 (AM1354) kit</td>
		</tr>
		<tr>
			<td>Phenol, Tris buffered</td>
			<td></td>
			<td></td>
			<td>High quality distilled phenol from any commercial supplier</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td></td>
			<td></td>
			<td>High quality chloroform from any commercial supplier</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>High quality ethanol from any commercial supplier</td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonate (DEPC)</td>
			<td>Sigma Chemical Co.</td>
			<td>D5758-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 protein, with nuclear localization signal</td>
			<td>PNA Bio, Inc.</td>
			<td>CP01</td>
			<td>Reconstituted using DEPC-treated water according to manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>10X Marc&#39;s Modified Ringers solution</td>
			<td></td>
			<td></td>
			<td>Homemade. Recipe (ref 26) for 1X MMR (lacking EDTA) is 100mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES. Solution is pH adjusted to 7.4.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td></td>
			<td></td>
			<td>Any Molecular Biology grade agarose is sufficient</td>
		</tr>
		<tr>
			<td>60 X15 mm Petri plates</td>
			<td>Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Falcon</td>
			<td>3047</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma Chemical Co.</td>
			<td>C7352-100G</td>
			<td>Free base, not HCl salt</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>03 115 828 001</td>
			<td>Typically ~20mg/ml from Roche</td>
		</tr>
		<tr>
			<td>sera Micron</td>
			<td>sera</td>
			<td></td>
			<td>Tadpole food. Resuspend in growth medium. Can be purchased from a variety of online retailers</td>
		</tr>
	</tbody>
</table>

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.

Following transplantation, the qualitative determination of the efficacy of CRISPR/Cas9 mutagenesis should be performed before expending the effort in animal husbandry to grow and maintain the animals to sexual maturity. Because animals carrying leapfrog transplants are somatically wildtype and the germline is difficult to access for direct measurements, saving the carcasses of donor embryos becomes important. DNA analysis from the carcasses serves as a proxy for the extent of mutagenesis...

Watch this Scientific Journal Video about Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus at JoVE.com

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.

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 ...

The overall goal of leapfrogging is to facilitate the creation of mutant lines for genes that are essential for embryogenesis. This method can help answer key questions in Developmental Genetics and other fields. The main advantage of this technique is that one can obtain mutants in essential genes in the F1 generation.To begin this procedure, microinject to Xenopus embryos at the one cell stage with Cas9-sgRNA complexes as outlined in the text protocol. When the embryos reach 4.5 hours post-fertilization, at early blastula stage nine, remove them from the 25 degree Celsius incubator to allow them to continue development at room temperature. To begin the transplantation at five hours post-fertilization, use a pasteur pipette to transfer one uninjected embryo to a 60 millimeter agarose dish containing depressions, which will serve as the graft recipient.Also transfer one PGC donor embryo to the dish. Use forceps to manually remove the vitelline envelopes from each embryo. And rotate both embryos so that their vegetal poles are in view and accessible for surgery.Next, while stabilizing the recipient embryo with the hair loop, insert the sharp tip of an eyebrow hair knife into the vegetal pole, just below the surface. And with rapid, upwards slicing movements, make four shallow incisions in the shape of a square, inside the zone where future bottle cells will form that mark the blastopore. Once the four sides of the square are delineated, use the eyebrow hair knife to deepen each incision using similar cutting motions to a depth of approximately 1/3 to 1/2 the distance to the blastocoel floor.Free the vegetal tissue explant from the recipient embryo by making one or more horizontal cuts in the deep vegetal region. Then, working quickly, repeat this procedure on the PGC donor embryo to create a similarly sized vegetal tissue fragment for transplantation. Once the tissue containing PGCs is removed from the donor embryo, use the eyebrow hair knife and hair loop to position the explant in the opening created in the recipient embryo.Use the shaft of the eyebrow hair knife, held parallel to the surface of the graft, to gently press the graft into the opening in the vegetal surface of the recipient embryo. Use the hair loop to gently slide the carcass of the donor embryo across the agarose surface and into a well. Make sure that the open wound of the carcass is facing the bulk liquid.Slide the recipient embryo into an adjacent well, and likewise, make sure that the grafted vegetal pole tissue is facing the bulk liquid. Repeat the transplantation of PGCs with additional embryos to create more transplant carcass pairs, until the embryos reach approximately early gastrula stage 10, transferring embryos to empty wells as the transplants are made. Once grafts have healed into place, typically 30 to 45 minutes after transplantation, use a pasteur pipette to very gently transfer embryos from the depressions to individual wells in an agarose-coated 24-well plate.Rotate the embryos so that they are positioned with the vegetal pole up, facing the bulk solution. Place the donor carcasses and graft recipients in adjacent wells to assist in keeping track of embryo pairs and record this information. Then, transfer the 24-well plate containing embryos to a 25 degree Celsius incubator overnight.The next day, move each graft recipient, now at the early tail bud stage, to an individual well of a fresh, agarose-coated, six-well plate. Raise the leapfrogged embryos and carry out analysis of donor DNA obtained from the carcasses according to the text protocol. Because leapfrogging produces animals that have efficient replacement of their germline with mutant alleles, mutant phenotypes can be obtained in the F1 generation.As presented in this table, Blitz et al. reported that a majority of F0 animals, targeted at the tyrosinase locus, which is required for melanocyte and retinal pigementation, show 100%germline transmission of donor-derived mutant genomes, as measured by outcrossing with tyr minus albino animals. Having watched this video, you should have a good understanding of how to transplant Xenopus primordial germ cells.

primordial-germ-cell-transplantation-for-crisprcas9-based

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMDMDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMDMDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMDMDX mutant mice.

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...