Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

Roose, Magdalena; Sauert, Kathrin; Turan, Gülüzar; Solomentsew, Natalie; Werdien, Dagmar; Pramanik, Kallal; Senkel, Sabine; Ryffel, Gerhart U.; Waldner, Christoph

doi:10.1007/s11248-009-9253-4

Cited by 17 publications

(17 citation statements)

References 48 publications

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“…backbone can be removed via the Cre/LoxP system (29,30). Taking the 5 founder frogs generating only GFP + /PCR 2 F1 progeny into consideration (i.e.…”

Section: Discussionmentioning

confidence: 99%

Heritable CRISPR/Cas9‐mediated targeted integration inXenopus tropicalis

et al. 2015

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Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/ Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.-Shi, Z., Wang, F., Cui, Y., Liu, Z., Guo, X., Zhang, Y., Deng, Y., Zhao, H., Chen, Y. Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis. FASEB J. 29, 4914-4923 (2015). www.fasebj.orgRecent establishment of efficient targeted gene disruption methods in Xenopus tropicalis (1-6) has confirmed this diploid frog as an excellent vertebrate genetic model; however, recombination-mediated genome editing has not been reported in this species.Direct injection of mRNAs of engineered nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), into fertilized eggs facilitates homologybased integration in zebrafish, mouse, and rat embryos (7-11). Unfortunately, taking similar strategies with either circular donor DNA, linear donor DNA, or synthetic oligodeoxynucleotides, we were unable to get heritable homologous recombination-mediated gene modification in X. tropicalis in the past several years, likely as a result of too low recombination efficiency.Here, we show that heritable targeted integration in X. tropicalis was successfully obtained when we took a homology-independent strategy recently developed for cell lines and zebrafish (12-15). The most prominent feature of the strategy is the introduction of a nuclease cleavage site into the donor DNA, thus allowing the concurrent cleavage of the chromosomal target site and circular donor DNA in vivo by a given nuclease. In addition, we designed a reporter system that helps identify early integration events. We tested the editing of 3 genes in X. tropicalis with this strategy. All showed efficient targeted integration that was passed to the next generation through germ-line transmission and was further confirmed by Southern blot analysis. Our data r...

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“…backbone can be removed via the Cre/LoxP system (29,30). Taking the 5 founder frogs generating only GFP + /PCR 2 F1 progeny into consideration (i.e.…”

Section: Discussionmentioning

confidence: 99%

Heritable CRISPR/Cas9‐mediated targeted integration inXenopus tropicalis

et al. 2015

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“…S1) to conditionally overexpress the P328L329del and A263insGG HNF1B mutants in developing Xenopus embryos as described previously [20]. The Cre recombinase gene in the HSPCre13 strain is linked to the red fluorescent tdTomato marker gene permitting easy identification of Cre positive animals (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the system has the advantage that the Cre recombinase is only transcribed after the heat shock minimizing the potential adverse effects of constitutive Cre expression. Previously, we have demonstrated that heat-shock induced overexpression of the P328L329del mutant causes pronephros malformations and large edemas in Xenopus tadpoles [20]. We now define the developmental stages responsive to HNF1B mutant overexpression and describe the pronephric defects by using well-defined molecular markers that define distinct domains of the pronephric nephron [15], [21].…”

Section: Introductionmentioning

confidence: 97%

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Heat-Shock Mediated Overexpression of HNF1β Mutations Has Differential Effects on Gene Expression in the Xenopus Pronephric Kidney

et al. 2012

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The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B.

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“…FLP and/or Cre systems have been developed to allow site-specific integration of transgenes into a variety of species including mice, fish, flies and even plants (Zou et al , 1994; Lyznik et al , 1996; Golic et al , 1997; Vergunst and Hooykaas, 1998; Vergunst et al , 1998; Horn and Handler, 2005; Oberstein et al , 2005; Shmerling et al , 2005; Liu et al , 2007). Although these enzymes can catalyze recombination in Xenopus , site-specific integration has not been reported (Werdien et al , 2001; Ryffel et al , 2003; Gargioli and Slack, 2004; Waldner et al , 2006; Rankin et al , 2009; Roose et al , 2009). The goal of this work was to determine if the FLP-FRT system could be used to perform recombinase-mediated cassette exchange in Xenopus .…”

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confidence: 99%

Site‐specific transgenesis in Xenopus

Zuber

Nihart

Zhuo

et al. 2012

Genesis

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Transgenesis is an essential, powerful tool for investigating gene function and the activities of enhancers, promoters and transcription factors in the chromatin environment. In Xenopus, current methods generate germ-line transgenics by random insertion, often resulting in mosaicism, position-dependent variations in expression, and lab-to-lab differences in efficiency. We have developed and tested a Xenopus FLP-FRT Recombinase Mediated Transgenesis (X-FRMT) method. We demonstrate transgenesis of Xenopus laevis by FLP-catalyzed recombination of donor plasmid cassettes into F1 tadpoles with host cassette transgenes. X-FRMT provides a new method for generating transgenic Xenopus. Once Xenopus lines harboring single host cassettes are generated, X-FRMT should allow for the targeting of transgenes to well characterized integration site(s), requiring no more special reagents or training than that already common to most Xenopus labs.

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Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

Cited by 17 publications

References 48 publications

Heritable CRISPR/Cas9‐mediated targeted integration inXenopus tropicalis

Heritable CRISPR/Cas9‐mediated targeted integration inXenopus tropicalis

Heat-Shock Mediated Overexpression of HNF1β Mutations Has Differential Effects on Gene Expression in the Xenopus Pronephric Kidney

Site‐specific transgenesis in Xenopus

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