Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ.

Duri Rungger,Lisbeth Muster,Elisabeth Rungger-Brändle,Oleg Georgiev

doi:10.1242/bio.022376

Duri Rungger, Lisbeth Muster + Show 2 more

Open Access

https://doi.org/10.1242/bio.022376

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Journal: Biology open	Publication Date: Feb 8, 2017
Citations: 2	License type: CC BY 3.0

Affiliation: University of Geneva, University of Zurich

Abstract

ABSTRACTThe newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance.

Highlights

In an earlier study (Hagmann et al, 1996), we tested the frequency of homologous recombination versus illegitimate end-to-end ligation using a linearized donor plasmid Reco-σ, which may undergo either end-to-end ligation yielding a plasmid conferring kanamycin resistance, or intramolecular homologous recombination producing a plasmid coding for tetracycline resistance
Transport of DNA from oocyte cytoplasm to nucleus To document the binding of biotinylated DNA by the core streptavidin (Sano et al, 1995) present in both shuttle proteins, gel retardation assays were performed using a synthetic oligonucleotide carrying a biotin at the 5′ end of one strand, and
Functional shuttle protein amounts to about 5% of our total protein preparation

Summary

Introduction

In an earlier study (Hagmann et al, 1996), we tested the frequency of homologous recombination versus illegitimate end-to-end ligation using a linearized donor plasmid Reco-σ, which may undergo either end-to-end ligation yielding a plasmid conferring kanamycin resistance, or intramolecular homologous recombination producing a plasmid coding for tetracycline resistance. The scope of the present study was to develop an oocyte shuttle protein (OS) that would transport donor DNA from the bloodstream into the oocyte nucleus within the ovary in situ, reaching a large number of oocytes in which homologous recombination takes place efficiently.

Results

Conclusion