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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.


Related in: MedlinePlus

Large scale expression of mCherry in sow milk.(a) Milk sample from a mCherry transgenic sow shown under specific excitation of mCherry (15 ms exposure). The milk cells were sedimented by centrifugation at the bottom of a 15 ml tube (concentrated out of 15 ml milk). Note the apparently higher expression of mCherry in the cells compared the skimmed milk. (b) Same sample shown under brightlight illumination. The high mCherry content colored the milk cells purple/reddish. (c) Skimmed milk fractions of milk from a mCherry (top) and a wildtype sow (bottom). At extended exposure times (here 500 ms), the mCherry could also be detected in the skimmed milk fraction. (d) Corresponding brightfield view of (c). (e) Top: Immunoblot of mCherry in protein fractions of milk cell extracts separated via an anion exchange column. M, size marker; -, empty slot; wt, wildtype milk cells; mCherry milk cells; F0–F 25, fractions 0 to 25. Arrow point to mCherry protein, migrating at an apparent molecular weight of 33 kD. Bottom: Corresponding Coomassie stained gel. (f) Absence of glycan-residues in Venus and mCherry proteins from sow milk. Immunodetection of Venus and mCherry. M, size marker; -, untreated milk proteins; P, PNGase treated; O, O-glycosidase/neuramidase treated; PO, PNGase and O-glycosidase/neuramidase treated. (g) Deglycosylation of fetuin control, and total milk proteins, same labels as in (f), Coomassie stained gel. (h) Absence of Venus and mCherry in blood plasma from transgenic sows, as determined by immunodetection. M, size marker; Pla, plasma; Ery, erythrocytes; Leu, leucocytes; V+, Venus positive milk sample; mCh+, mCherry positive milk sample. (i) Corresponding Coomassie stained gel of (h).
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f4: Large scale expression of mCherry in sow milk.(a) Milk sample from a mCherry transgenic sow shown under specific excitation of mCherry (15 ms exposure). The milk cells were sedimented by centrifugation at the bottom of a 15 ml tube (concentrated out of 15 ml milk). Note the apparently higher expression of mCherry in the cells compared the skimmed milk. (b) Same sample shown under brightlight illumination. The high mCherry content colored the milk cells purple/reddish. (c) Skimmed milk fractions of milk from a mCherry (top) and a wildtype sow (bottom). At extended exposure times (here 500 ms), the mCherry could also be detected in the skimmed milk fraction. (d) Corresponding brightfield view of (c). (e) Top: Immunoblot of mCherry in protein fractions of milk cell extracts separated via an anion exchange column. M, size marker; -, empty slot; wt, wildtype milk cells; mCherry milk cells; F0–F 25, fractions 0 to 25. Arrow point to mCherry protein, migrating at an apparent molecular weight of 33 kD. Bottom: Corresponding Coomassie stained gel. (f) Absence of glycan-residues in Venus and mCherry proteins from sow milk. Immunodetection of Venus and mCherry. M, size marker; -, untreated milk proteins; P, PNGase treated; O, O-glycosidase/neuramidase treated; PO, PNGase and O-glycosidase/neuramidase treated. (g) Deglycosylation of fetuin control, and total milk proteins, same labels as in (f), Coomassie stained gel. (h) Absence of Venus and mCherry in blood plasma from transgenic sows, as determined by immunodetection. M, size marker; Pla, plasma; Ery, erythrocytes; Leu, leucocytes; V+, Venus positive milk sample; mCh+, mCherry positive milk sample. (i) Corresponding Coomassie stained gel of (h).

Mentions: Subsequently, the milk from three mCherry transposon sows was analyzed. In the milk cell fractions, the mCherry fluorophore was readily detectable (Fig. 4), actually the high mCherry content colored the milk cells (here concentrated from 15 ml milk) reddish under white light illumination. The skimmed milk fraction apparently contained lower mCherry concentrations than the pellet of the milk cells (Fig. 4), supporting the notion that indeed the somatic milk cells carried the mCherry protein in a piggyback manner into the milk. The mCherry from extracted milk cells could be enriched via column purification and expression could be confirmed by immunoblotting with an anti-mCherry antibody (Fig. 4). The content of recombinant mCherry protein in milk from the transgenic sows was determined to be 0.20–0.25 g/l (Fig. 3c,d).


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)

Large scale expression of mCherry in sow milk.(a) Milk sample from a mCherry transgenic sow shown under specific excitation of mCherry (15 ms exposure). The milk cells were sedimented by centrifugation at the bottom of a 15 ml tube (concentrated out of 15 ml milk). Note the apparently higher expression of mCherry in the cells compared the skimmed milk. (b) Same sample shown under brightlight illumination. The high mCherry content colored the milk cells purple/reddish. (c) Skimmed milk fractions of milk from a mCherry (top) and a wildtype sow (bottom). At extended exposure times (here 500 ms), the mCherry could also be detected in the skimmed milk fraction. (d) Corresponding brightfield view of (c). (e) Top: Immunoblot of mCherry in protein fractions of milk cell extracts separated via an anion exchange column. M, size marker; -, empty slot; wt, wildtype milk cells; mCherry milk cells; F0–F 25, fractions 0 to 25. Arrow point to mCherry protein, migrating at an apparent molecular weight of 33 kD. Bottom: Corresponding Coomassie stained gel. (f) Absence of glycan-residues in Venus and mCherry proteins from sow milk. Immunodetection of Venus and mCherry. M, size marker; -, untreated milk proteins; P, PNGase treated; O, O-glycosidase/neuramidase treated; PO, PNGase and O-glycosidase/neuramidase treated. (g) Deglycosylation of fetuin control, and total milk proteins, same labels as in (f), Coomassie stained gel. (h) Absence of Venus and mCherry in blood plasma from transgenic sows, as determined by immunodetection. M, size marker; Pla, plasma; Ery, erythrocytes; Leu, leucocytes; V+, Venus positive milk sample; mCh+, mCherry positive milk sample. (i) Corresponding Coomassie stained gel of (h).
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Related In: Results  -  Collection

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f4: Large scale expression of mCherry in sow milk.(a) Milk sample from a mCherry transgenic sow shown under specific excitation of mCherry (15 ms exposure). The milk cells were sedimented by centrifugation at the bottom of a 15 ml tube (concentrated out of 15 ml milk). Note the apparently higher expression of mCherry in the cells compared the skimmed milk. (b) Same sample shown under brightlight illumination. The high mCherry content colored the milk cells purple/reddish. (c) Skimmed milk fractions of milk from a mCherry (top) and a wildtype sow (bottom). At extended exposure times (here 500 ms), the mCherry could also be detected in the skimmed milk fraction. (d) Corresponding brightfield view of (c). (e) Top: Immunoblot of mCherry in protein fractions of milk cell extracts separated via an anion exchange column. M, size marker; -, empty slot; wt, wildtype milk cells; mCherry milk cells; F0–F 25, fractions 0 to 25. Arrow point to mCherry protein, migrating at an apparent molecular weight of 33 kD. Bottom: Corresponding Coomassie stained gel. (f) Absence of glycan-residues in Venus and mCherry proteins from sow milk. Immunodetection of Venus and mCherry. M, size marker; -, untreated milk proteins; P, PNGase treated; O, O-glycosidase/neuramidase treated; PO, PNGase and O-glycosidase/neuramidase treated. (g) Deglycosylation of fetuin control, and total milk proteins, same labels as in (f), Coomassie stained gel. (h) Absence of Venus and mCherry in blood plasma from transgenic sows, as determined by immunodetection. M, size marker; Pla, plasma; Ery, erythrocytes; Leu, leucocytes; V+, Venus positive milk sample; mCh+, mCherry positive milk sample. (i) Corresponding Coomassie stained gel of (h).
Mentions: Subsequently, the milk from three mCherry transposon sows was analyzed. In the milk cell fractions, the mCherry fluorophore was readily detectable (Fig. 4), actually the high mCherry content colored the milk cells (here concentrated from 15 ml milk) reddish under white light illumination. The skimmed milk fraction apparently contained lower mCherry concentrations than the pellet of the milk cells (Fig. 4), supporting the notion that indeed the somatic milk cells carried the mCherry protein in a piggyback manner into the milk. The mCherry from extracted milk cells could be enriched via column purification and expression could be confirmed by immunoblotting with an anti-mCherry antibody (Fig. 4). The content of recombinant mCherry protein in milk from the transgenic sows was determined to be 0.20–0.25 g/l (Fig. 3c,d).

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.


Related in: MedlinePlus