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Genesis
2017 Jan 01;551-2:. doi: 10.1002/dvg.23006.
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Targeted integration of genes in Xenopus tropicalis.
Shi Z
,
Tian D
,
Xin H
,
Lian J
,
Guo X
,
Chen Y
.
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With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.
Figure 1.
TALEN-mediated homology-dependent gene targeting strategy in X. tropicalis resulted in about 2% of injected tadpoles expressing the selection marker, in which HR-mediated targeted integration can be detected by PCR analysis. (a) Schematic of HR-mediated targeting at X. tropicalis ets1 locus with a circular donor DNA. TALEN mRNAs (500 pg/embryo) and circular donor DNA (20 pg/embryo) in 2 nl of volume was co-injected into 1-cell stage X. tropicalis embryos from the animal pole. GFP expression was inspected and photographed at stages 42–46 of development with a fluorescence stereomicroscope equipped with a CCD camera (Olympus, Tokyo, Japan). The pancreas-green tadpoles were sorted out for further PCR and germ line transmission analyses. Sequences on the top illustrate the effective TALEN targeting site (Lei et al., 2012) with TALEN binding sequences in orange. Red arrows indicate the location of primers for amplifying the two integration junctions. E, exon; LA, left homology arm, 1.2 kb; RA, right homology arm, 1.2 kb; pA, SV40 polyadenylation signal. (b) A representative stage 45 tadpole showed mosaic pancreatic GFP (white arrow). The bright spot on the left is the autofluorescence from gallbladder. Scale bar, 200 µm. (c) PCR assay data revealed expected integration at the right homology arm in sorted individual GFP+ tadpoles. M, molecular marker; NC, negative control; wt, wild type
Figure 2.
Schematic of homology-independent strategy for targeted integration in X. tropicalis. Upon coinjection of Cas9 mRNA, gRNA, and circular donor DNA into X. tropicalis fertilized eggs at animal pole, chromosomal target site and donor plasmid would be simultaneously cleaved in vivo. Resultant DNA ends would be ligated by error-prone nonhomologous repairs (NHRs). If desired, in principle, the vector backbone and the selection cassette (ela:EGFP) can be removed with the Cre/LoxP system. GOI, gene of interest; pA, SV40 polyadenylation signal
