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Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway.

Mukherjee A, Garrels W, Talluri TR, Tiedemann D, Bősze Z, Ivics Z, Kues WA - Sci Rep (2016)

Bottom Line: Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found.A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk.This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder.

View Article: PubMed Central - PubMed

Affiliation: Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Mariensee, Germany.

ABSTRACT
We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder.

No MeSH data available.


Determination of fluorophore concentrations in milk of transgenic sows.(a) Different concentrations of a recombinant GFP-fusion protein (58kD) were loaded alongside of milk samples from a wildtype (wt) and a Venus transposon sow (#535) on a 12% SDS-PAGE, blotted to a PVDF membrane, and probed with an anti-EGFP antibody. (b) Densitometric analysis of blot suggesting a Venus concentration of 0,27–0.38 g/l in milk from transgenic sows. (c) Different concentrations of a recombinant mCherry protein were loaded alongside of milk samples from a wildtype (wt) and a mCherry transposon sow (#594) on a 12% SDS-PAGE, blotted to a PVDF membrane, probed with an anti-mCherry antibody, and densitometrically evaluated. (d) Densitometric analysis suggesting a mCherry concentration of 0.20–0.25 g/l in milk from transgenic sows.
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f3: Determination of fluorophore concentrations in milk of transgenic sows.(a) Different concentrations of a recombinant GFP-fusion protein (58kD) were loaded alongside of milk samples from a wildtype (wt) and a Venus transposon sow (#535) on a 12% SDS-PAGE, blotted to a PVDF membrane, and probed with an anti-EGFP antibody. (b) Densitometric analysis of blot suggesting a Venus concentration of 0,27–0.38 g/l in milk from transgenic sows. (c) Different concentrations of a recombinant mCherry protein were loaded alongside of milk samples from a wildtype (wt) and a mCherry transposon sow (#594) on a 12% SDS-PAGE, blotted to a PVDF membrane, probed with an anti-mCherry antibody, and densitometrically evaluated. (d) Densitometric analysis suggesting a mCherry concentration of 0.20–0.25 g/l in milk from transgenic sows.

Mentions: The skimmed milk fraction from Venus transposon sows could be used to enrich the Venus protein by size chromatography (Fig. 2, Supplementary Fig. S1). The content of recombinant Venus protein was determined to be between 0.27–0.38 g/l of milk (Fig. 3).


Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway.

Mukherjee A, Garrels W, Talluri TR, Tiedemann D, Bősze Z, Ivics Z, Kues WA - Sci Rep (2016)

Determination of fluorophore concentrations in milk of transgenic sows.(a) Different concentrations of a recombinant GFP-fusion protein (58kD) were loaded alongside of milk samples from a wildtype (wt) and a Venus transposon sow (#535) on a 12% SDS-PAGE, blotted to a PVDF membrane, and probed with an anti-EGFP antibody. (b) Densitometric analysis of blot suggesting a Venus concentration of 0,27–0.38 g/l in milk from transgenic sows. (c) Different concentrations of a recombinant mCherry protein were loaded alongside of milk samples from a wildtype (wt) and a mCherry transposon sow (#594) on a 12% SDS-PAGE, blotted to a PVDF membrane, probed with an anti-mCherry antibody, and densitometrically evaluated. (d) Densitometric analysis suggesting a mCherry concentration of 0.20–0.25 g/l in milk from transgenic sows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834472&req=5

f3: Determination of fluorophore concentrations in milk of transgenic sows.(a) Different concentrations of a recombinant GFP-fusion protein (58kD) were loaded alongside of milk samples from a wildtype (wt) and a Venus transposon sow (#535) on a 12% SDS-PAGE, blotted to a PVDF membrane, and probed with an anti-EGFP antibody. (b) Densitometric analysis of blot suggesting a Venus concentration of 0,27–0.38 g/l in milk from transgenic sows. (c) Different concentrations of a recombinant mCherry protein were loaded alongside of milk samples from a wildtype (wt) and a mCherry transposon sow (#594) on a 12% SDS-PAGE, blotted to a PVDF membrane, probed with an anti-mCherry antibody, and densitometrically evaluated. (d) Densitometric analysis suggesting a mCherry concentration of 0.20–0.25 g/l in milk from transgenic sows.
Mentions: The skimmed milk fraction from Venus transposon sows could be used to enrich the Venus protein by size chromatography (Fig. 2, Supplementary Fig. S1). The content of recombinant Venus protein was determined to be between 0.27–0.38 g/l of milk (Fig. 3).

Bottom Line: Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found.A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk.This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder.

View Article: PubMed Central - PubMed

Affiliation: Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Mariensee, Germany.

ABSTRACT
We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder.

No MeSH data available.