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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.


In silico prediction of signal peptides.(a) Design of transposons. The CAGGS promoter driven cDNA is flanked by heterospecific loxP sites and the SB inverted terminal repeats (ITR). Here the Venus transposon is depicted, the mCherry transposon has an identical design. (b,c) Signal peptide analysis of the Venus and mCherry coding sequences. Note, that the algorithm does not predict a signal peptide for Venus or for mCherry. (d,e) Signal peptide analysis of porcine alpha s1 (GenBank: EU025875.1) and beta caseins (GenBank: EU213063.1). Note, the distinct prediction of signal peptides in the milk proteins. C-score, raw cleavage site score; S-score, signal peptide score; Y-score, combined cleavage site score. For further details see SignalP4.1 website (www. http://www.cbs.dtu.dk/services/SignalP/output.php).
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f1: In silico prediction of signal peptides.(a) Design of transposons. The CAGGS promoter driven cDNA is flanked by heterospecific loxP sites and the SB inverted terminal repeats (ITR). Here the Venus transposon is depicted, the mCherry transposon has an identical design. (b,c) Signal peptide analysis of the Venus and mCherry coding sequences. Note, that the algorithm does not predict a signal peptide for Venus or for mCherry. (d,e) Signal peptide analysis of porcine alpha s1 (GenBank: EU025875.1) and beta caseins (GenBank: EU213063.1). Note, the distinct prediction of signal peptides in the milk proteins. C-score, raw cleavage site score; S-score, signal peptide score; Y-score, combined cleavage site score. For further details see SignalP4.1 website (www. http://www.cbs.dtu.dk/services/SignalP/output.php).

Mentions: A bioinformatic analysis predicted no secretion signal sequence in the Venus or mCherry sequences (Fig. 1), whereas a clear signal peptide prediction was obtained for known porcine milk proteins, like alpha s1 casein and beta casein. Nevertheless, milk samples collected from lactating transposon-transgenic sows contained high levels of the respective recombinant reporter proteins, which could be readily identified by fluorescence microscopy (Fig. 2). In total, milk samples were collected from two Venus transposon sows, three mCherry transposon sows and five control (non-transgenic) sows.


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)

In silico prediction of signal peptides.(a) Design of transposons. The CAGGS promoter driven cDNA is flanked by heterospecific loxP sites and the SB inverted terminal repeats (ITR). Here the Venus transposon is depicted, the mCherry transposon has an identical design. (b,c) Signal peptide analysis of the Venus and mCherry coding sequences. Note, that the algorithm does not predict a signal peptide for Venus or for mCherry. (d,e) Signal peptide analysis of porcine alpha s1 (GenBank: EU025875.1) and beta caseins (GenBank: EU213063.1). Note, the distinct prediction of signal peptides in the milk proteins. C-score, raw cleavage site score; S-score, signal peptide score; Y-score, combined cleavage site score. For further details see SignalP4.1 website (www. http://www.cbs.dtu.dk/services/SignalP/output.php).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: In silico prediction of signal peptides.(a) Design of transposons. The CAGGS promoter driven cDNA is flanked by heterospecific loxP sites and the SB inverted terminal repeats (ITR). Here the Venus transposon is depicted, the mCherry transposon has an identical design. (b,c) Signal peptide analysis of the Venus and mCherry coding sequences. Note, that the algorithm does not predict a signal peptide for Venus or for mCherry. (d,e) Signal peptide analysis of porcine alpha s1 (GenBank: EU025875.1) and beta caseins (GenBank: EU213063.1). Note, the distinct prediction of signal peptides in the milk proteins. C-score, raw cleavage site score; S-score, signal peptide score; Y-score, combined cleavage site score. For further details see SignalP4.1 website (www. http://www.cbs.dtu.dk/services/SignalP/output.php).
Mentions: A bioinformatic analysis predicted no secretion signal sequence in the Venus or mCherry sequences (Fig. 1), whereas a clear signal peptide prediction was obtained for known porcine milk proteins, like alpha s1 casein and beta casein. Nevertheless, milk samples collected from lactating transposon-transgenic sows contained high levels of the respective recombinant reporter proteins, which could be readily identified by fluorescence microscopy (Fig. 2). In total, milk samples were collected from two Venus transposon sows, three mCherry transposon sows and five control (non-transgenic) sows.

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.