XB-ART-52054Dev Biol. January 1, 2017; 426 (2): 325-335.
Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling.
The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.
PubMed ID: 27109192
PMC ID: PMC5074924
Article link: Dev Biol.
Grant support: P40 OD010997 NIH HHS , R01 HD084409 NICHD NIH HHS , R01 HL112618 NHLBI NIH HHS , R01 HL127640 NHLBI NIH HHS
Genes referenced: kidins220
Article Images: [+] show captions
|Fig. 1. Genome editing using Xenopus embryos or oocytes. A comparison of the steps required to generate Xenopus mutants using either embryo injection or oocyte-host transfer methods. (A) For the embryo injection method, TALEN mRNAs or Cas9 mRNA and/or protein along with sgRNA is microinjected into fertilized embryos at the one-cell stage. The embryos are genotyped to confirm editing efficiency using PCR-sequencing, T7 endonuclease assays, or high-resolution melt analysis. The F0 mosaic embryos are allowed to develop, and gene function is analyzed using a host of established assays, including in situ hybridization (ISH) and immunohistochemistry (IHC). These embryos, once grown to adulthood, can be tested for germline transmission to generate subsequent mutant lines. (B) An image of a mutant frog generated from embryos injected at the one-cell stage with TALEN mRNAs targeting the tyrosinase gene shows mosaic pigmentation throughout the skin. (C) For the oocyte-host transfer method, stage VI oocytes are surgically removed from an adult female frog, manually defollicated, and microinjected with TALENs or CRISPR-Cas9 capped mRNA and/or protein. The oocytes are then matured using progesterone and colored with vital dyes for visualization; the coloring of oocytes is not necessary if implanted into an albino female. The oocytes are then transferred into pre-primed host females and subsequently laid to incorporate the jelly coat that is essential for in vitro fertilization with sperm. The resulting embryos are genotyped and phenotyped as previously described. (D) An image of a mutant frog generated from oocytes injected with the same TALENs as in panel B, targeting the tyrosinase gene, shows more dramatic levels of albinism than the embryo-injected frog, thereby confirming more efficient mutagenesis.|
|Fig. 2. Integrating exogenous DNA into Xenopus using genetic editing tools. Outline of the various knock-in strategies that have been employed to insert DNA into a targeted genomic locus in Xenopus. (A) Nakade et al. described the use of TALENs and microhomology-mediated end joining (MMEJ, TAL-PITCh) to integrate a fluorescent protein (eg. GFP) at the end of the coding region 5′ to the endogenous stop codon. (B) Shi et al. utilized CRISPR-Cas editing to insert plasmid DNA harboring a known pancreas tissue enhancer element (Elastase promoter) driving GFP, into the intron of their target gene. (C) Jaffe et al., used targeting constructs containing allele-specific homology arms to insert fluorescent proteins into a sgRNA-targeted exon, thereby visualizing cells in which specific gene function was abrogated. TAA; stop codon, FokI; Fok1 nuclease, GFP; green fluorescent protein, pA; poly-A tail, sgRNA; guide RNA for CRISPR.|
|Fig. 3. Workflow for generating Xenopus mutants using TALENs and CRISPR-Cas9. A schematic depicts the steps required to generate the gene editing tools to target a gene of interest, induce mutations in Xenopus embryos, perform subsequent assays to phenotype mosaic F0 embryos, and generate mutant lines. For more detailed information including web URLs we refer the reader to Xenbase (http://www.xenbase.org/other/static/CRISPr.jsp).|