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BACKGROUND: Xenopus embryos and tadpoles are versatile models for embryological, cell biological, and regenerative studies. Genomic and transcriptomic approaches have been increasingly employed in these frogs. Most of these genome-wide analyses have profiled tissues in bulk, but there are many scenarios where isolation of single cells may be advantageous, including isolation of a preferred cell type, or generation of a single-cell suspension for applications such as scRNA-Seq.
RESULTS: Here we present a protocol for the disaggregation of complex tail and limb bud tissue, and use cell type-specific fluorescence in transgenic X. tropicalis appendages to isolate specific cell populations using fluorescence activated cell sorting (FACS). Our protocol addresses a specific challenge in Xenopus embryos and tadpoles: the storage of maternal yolk platelets in each cell, which can introduce light scatter and thereby false positives into FACS analysis.
CONCLUSIONS: Here we gate against both nontransgenic and ubiquitously transgenic animals to reduce both false positives and false negatives. We use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP)Papal transgenic line as a test case to demonstrate that nucleic acid preparations made from sorted cells are high quality and specific. We anticipate this method will be adaptable to study various cell types that have transgenic reporter lines to better profile cell types of interest.
Aztekin,
Identification of a regeneration-organizing cell in the Xenopus tail.
2019, Pubmed,
Xenbase
Aztekin,
Identification of a regeneration-organizing cell in the Xenopus tail.
2019,
Pubmed
,
Xenbase
Beck,
Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms.
2009,
Pubmed
,
Xenbase
Beliakova-Bethell,
The effect of cell subset isolation method on gene expression in leukocytes.
2014,
Pubmed
Binek,
Flow Cytometry Has a Significant Impact on the Cellular Metabolome.
2019,
Pubmed
Bonner,
Fluorescence activated cell sorting.
1972,
Pubmed
Briggs,
The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution.
2018,
Pubmed
,
Xenbase
Cerda,
RNA profiling of FAC-sorted neurons from the developing zebrafish spinal cord.
2009,
Pubmed
Dhorne-Pollet,
Validation of novel reference genes for RT-qPCR studies of gene expression in Xenopus tropicalis during embryonic and post-embryonic development.
2013,
Pubmed
,
Xenbase
Duncan,
Xenopus as a model organism for birth defects-Congenital heart disease and heterotaxy.
2016,
Pubmed
,
Xenbase
Gallardo,
Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells.
2013,
Pubmed
Gerber,
Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.
2018,
Pubmed
Gilbert,
Temporal order of replication of Xenopus laevis 5S ribosomal RNA genes in somatic cells.
1986,
Pubmed
,
Xenbase
Godsave,
Clonal analysis of mesoderm induction in Xenopus laevis.
1989,
Pubmed
,
Xenbase
Grainger,
Xenopus tropicalis as a model organism for genetics and genomics: past, present, and future.
2012,
Pubmed
,
Xenbase
Hadjantonakis,
FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter.
2000,
Pubmed
Harland,
Xenopus research: metamorphosed by genetics and genomics.
2011,
Pubmed
,
Xenbase
Hartley,
Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.
2001,
Pubmed
,
Xenbase
Hirsch,
Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.
2002,
Pubmed
,
Xenbase
Horb,
Xenopus Resources: Transgenic, Inbred and Mutant Animals, Training Opportunities, and Web-Based Support.
2019,
Pubmed
,
Xenbase
Johnson,
pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos.
1994,
Pubmed
,
Xenbase
Kakebeen,
Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis.
2019,
Pubmed
,
Xenbase
Kakebeen,
More Than Just a Bandage: Closing the Gap Between Injury and Appendage Regeneration.
2019,
Pubmed
,
Xenbase
Kakebeen,
Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors.
2020,
Pubmed
,
Xenbase
Llufrio,
Sorting cells alters their redox state and cellular metabolome.
2018,
Pubmed
Manoli,
Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.
2012,
Pubmed
Nenni,
Xenbase: Facilitating the Use of Xenopus to Model Human Disease.
2019,
Pubmed
,
Xenbase
Noelanders,
Cell Cycle Analysis of the Embryonic Brain of Fluorescent Reporter Xenopus tropicalis by Flow Cytometry.
2018,
Pubmed
,
Xenbase
Ny,
A transgenic Xenopus laevis reporter model to study lymphangiogenesis.
2013,
Pubmed
,
Xenbase
Phipps,
Model systems for regeneration: Xenopus.
2020,
Pubmed
,
Xenbase
Raudvere,
g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update).
2019,
Pubmed
Reichard,
Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry.
2019,
Pubmed
Richardson,
Does FACS perturb gene expression?
2015,
Pubmed
Robinson,
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
2010,
Pubmed
Sargent,
Cell interactions and the control of gene activity during early development of Xenopus laevis.
1986,
Pubmed
,
Xenbase
Sato,
Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange.
2018,
Pubmed
,
Xenbase
Selman,
The utilization of yolk platelets by tissues of Xenopus embryos studied by a safranin staining method.
1965,
Pubmed
,
Xenbase
Suzuki,
Limb regeneration in Xenopus laevis froglet.
2006,
Pubmed
,
Xenbase
Tsujioka,
Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.
2015,
Pubmed
,
Xenbase
