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The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis.

Abu-Daya A, Nishimoto S, Fairclough L, Mohun TJ, Logan MP, Zimmerman LB - Dev. Biol. (2010)

Bottom Line: While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans.Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis.Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.

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Sequences at either end of the xdm insertion were obtained by LM-PCR (5′ junction of insertion with respect to npnt transcript) or by PCR with transgene- and scaffold 111-specific primers (3′ end). Sequence corresponding to X. tropicalis v.4.1 scaffold_111 is shown in blue while sequence of the X. laevis Nkx2.5 promoter is in red; polylinker sequence from the transgene vector is in green; and sequence of unknown origin is in black. Sequences are numbered with respect to the start of scaffold_111 and the Nkx2.5b promoter (Genbank accession number: GU573788).
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f0045: Sequences at either end of the xdm insertion were obtained by LM-PCR (5′ junction of insertion with respect to npnt transcript) or by PCR with transgene- and scaffold 111-specific primers (3′ end). Sequence corresponding to X. tropicalis v.4.1 scaffold_111 is shown in blue while sequence of the X. laevis Nkx2.5 promoter is in red; polylinker sequence from the transgene vector is in green; and sequence of unknown origin is in black. Sequences are numbered with respect to the start of scaffold_111 and the Nkx2.5b promoter (Genbank accession number: GU573788).

Mentions: To characterize the transgene locus, we used Ligation-Mediated PCR (LM-PCR) with primers specific to both ends of the transgene to locate the integration at bp 91378–91352 in scaffold_111 of the JGI X. tropicalis genome assembly (http://www.genome.jgi-psf.org/Xentr4/Xentr4.home.html) in the third intron of the nephronectin (npnt) gene (Fig. 3A). Concatameric transgene insertions often result when Xenopus eggs are injected with sperm nuclei incubated with linear DNA constructs (Kroll and Amaya, 1996). Only 26 bp of endogenous npnt intron sequence is missing in the mutant allele, consistent with a relatively clean transgene insertion (Figs. 3A and S1).


The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis.

Abu-Daya A, Nishimoto S, Fairclough L, Mohun TJ, Logan MP, Zimmerman LB - Dev. Biol. (2010)

Sequences at either end of the xdm insertion were obtained by LM-PCR (5′ junction of insertion with respect to npnt transcript) or by PCR with transgene- and scaffold 111-specific primers (3′ end). Sequence corresponding to X. tropicalis v.4.1 scaffold_111 is shown in blue while sequence of the X. laevis Nkx2.5 promoter is in red; polylinker sequence from the transgene vector is in green; and sequence of unknown origin is in black. Sequences are numbered with respect to the start of scaffold_111 and the Nkx2.5b promoter (Genbank accession number: GU573788).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3021715&req=5

f0045: Sequences at either end of the xdm insertion were obtained by LM-PCR (5′ junction of insertion with respect to npnt transcript) or by PCR with transgene- and scaffold 111-specific primers (3′ end). Sequence corresponding to X. tropicalis v.4.1 scaffold_111 is shown in blue while sequence of the X. laevis Nkx2.5 promoter is in red; polylinker sequence from the transgene vector is in green; and sequence of unknown origin is in black. Sequences are numbered with respect to the start of scaffold_111 and the Nkx2.5b promoter (Genbank accession number: GU573788).
Mentions: To characterize the transgene locus, we used Ligation-Mediated PCR (LM-PCR) with primers specific to both ends of the transgene to locate the integration at bp 91378–91352 in scaffold_111 of the JGI X. tropicalis genome assembly (http://www.genome.jgi-psf.org/Xentr4/Xentr4.home.html) in the third intron of the nephronectin (npnt) gene (Fig. 3A). Concatameric transgene insertions often result when Xenopus eggs are injected with sperm nuclei incubated with linear DNA constructs (Kroll and Amaya, 1996). Only 26 bp of endogenous npnt intron sequence is missing in the mutant allele, consistent with a relatively clean transgene insertion (Figs. 3A and S1).

Bottom Line: While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans.Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis.Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.

Show MeSH