Volume 57, Issue 10 e23329

TECHNOLOGY REPORT

Novel vectors for functional interrogation of Xenopus ORFeome coding sequences

Zachary R. Sterner,

Zachary R. Sterner

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

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Scott A. Rankin,

Scott A. Rankin

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Marcin Wlizla,

Marcin Wlizla

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Jinyoung A. Choi,

Jinyoung A. Choi

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

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David M. Luedeke,

David M. Luedeke

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Aaron M. Zorn,

Aaron M. Zorn

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Daniel R. Buchholz,

Corresponding Author

Daniel R. Buchholz

buchhodr@ucmail.uc.edu

orcid.org/0000-0003-2070-5684

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

Correspondence

Daniel R. Buchholz, Department of Biological Sciences, University of Cincinnati, Cincinnati, OH.

Email: buchhodr@ucmail.uc.edu

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Zachary R. Sterner,

Zachary R. Sterner

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

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Scott A. Rankin,

Scott A. Rankin

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

Search for more papers by this author

Marcin Wlizla,

Marcin Wlizla

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Jinyoung A. Choi,

Jinyoung A. Choi

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

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David M. Luedeke,

David M. Luedeke

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Aaron M. Zorn,

Aaron M. Zorn

Division of Developmental Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

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Daniel R. Buchholz,

Corresponding Author

Daniel R. Buchholz

buchhodr@ucmail.uc.edu

orcid.org/0000-0003-2070-5684

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

Correspondence

Daniel R. Buchholz, Department of Biological Sciences, University of Cincinnati, Cincinnati, OH.

Email: buchhodr@ucmail.uc.edu

Search for more papers by this author

First published: 12 August 2019

https://doi.org/10.1002/dvg.23329

Citations: 3

Present address: Marcin Wlizla, National Xenopus Resource, Marine Biological Laboratory, Woods Hole, MA.

Jinyoung A. Choi, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Weill Cornell Medical College, New York, NY.

Funding information: National Institutes of Health, Grant/Award Number: P01HD093363

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Abstract

The current Xenopus ORFeome contains ~10,250 validated, full-length cDNA sequences without stop codons from Xenopus laevis and ~3,970 from Xenopus tropicalis cloned into Gateway-compatible entry vectors. To increase the utility of the ORFeome, we have constructed the Gateway-compatible destination vectors pDXTP and pDXTR, which in combination can control the spatial and temporal expression of any open reading frame (ORF). pDXTP receives a promoter/enhancer of interest, which controls the spatial expression of a doxycycline-inducible transcription factor rtTA. pDXTR receives an ORF of interest, which is controlled by a tetracycline response element enabling temporal control of ORF expression via rtTA activation by simple addition of doxycycline to the rearing water at any desired time point. These vectors can be integrated into the genome via well-established microinjection-based SceI, tol2, or phi-C31 transgenesis procedures and contain fluorescence reporters to confirm transgene integration. Cell-autonomous verification of ORF expression occurs via red nuclear fluorescence due to an mCherry-histone H2B fusion protein that is cleaved from the ORF during translation. Function of all essential features of pDXTP and pDXTR has been experimentally validated. pDXTP and pDXTR provide flexible molecular cloning and transgenesis options to accomplish tissue-specific inducible control of ORF expression in transgenic Xenopus.

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Volume57, Issue10

October 2019

e23329

Novel vectors for functional interrogation of Xenopus ORFeome coding sequences

Abstract

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Novel vectors for functional interrogation of Xenopus ORFeome coding sequences

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