Limits...
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.


Time course of reporter expression during lactation.(a) Expression of mCherry in somatic cells and (b) skimmed milk, collected from colostrum (cd1), and at days 3, 6, 12, 26 and 33 (d3-d33) of lactation; M, size marker; cc, control colostrum from wildtype sow; neg., negative milk control; pos, positive milk control (from mCherry sow). Left immunoblot with anti-mCherry antibody, and right corresponding Coomasssie stained gel (loading control). Note that the colostrum of the transgenic sow (cd1) did not show detectable mCherry expression. In the somatic cells the mCherry signal comes up at d3 (arrow), and in the skimmed milk at day 6 (arrow). The signal intensity is normalized to the marker bands and the positive control, suggesting that the majority of mCherry is deposited in the somatic cells, and released to the skimmed milk by membrane-damaged cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4834472&req=5

f5: Time course of reporter expression during lactation.(a) Expression of mCherry in somatic cells and (b) skimmed milk, collected from colostrum (cd1), and at days 3, 6, 12, 26 and 33 (d3-d33) of lactation; M, size marker; cc, control colostrum from wildtype sow; neg., negative milk control; pos, positive milk control (from mCherry sow). Left immunoblot with anti-mCherry antibody, and right corresponding Coomasssie stained gel (loading control). Note that the colostrum of the transgenic sow (cd1) did not show detectable mCherry expression. In the somatic cells the mCherry signal comes up at d3 (arrow), and in the skimmed milk at day 6 (arrow). The signal intensity is normalized to the marker bands and the positive control, suggesting that the majority of mCherry is deposited in the somatic cells, and released to the skimmed milk by membrane-damaged cells.

Mentions: A time-course experiment to follow the expression of the reporter proteins from day 1 of lactation (colostrum) to weaning is shown in Fig. 5. Interestingly, in colostrum no expression of the reporter (mCherry) could be detected. The mCherry expression started in the somatic cells from day 3 and was then detectable at high levels until weaning. In the skimmed milk, expression of mCherry was first detectable at day 6 and was then continuously present until weaning, but in lower levels than in the somatic cells (Fig. 5). The total amounts of exfoliated cells were similar in the milk from transgenic and control wildtype sows (Supplementary Fig. S2).


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)

Time course of reporter expression during lactation.(a) Expression of mCherry in somatic cells and (b) skimmed milk, collected from colostrum (cd1), and at days 3, 6, 12, 26 and 33 (d3-d33) of lactation; M, size marker; cc, control colostrum from wildtype sow; neg., negative milk control; pos, positive milk control (from mCherry sow). Left immunoblot with anti-mCherry antibody, and right corresponding Coomasssie stained gel (loading control). Note that the colostrum of the transgenic sow (cd1) did not show detectable mCherry expression. In the somatic cells the mCherry signal comes up at d3 (arrow), and in the skimmed milk at day 6 (arrow). The signal intensity is normalized to the marker bands and the positive control, suggesting that the majority of mCherry is deposited in the somatic cells, and released to the skimmed milk by membrane-damaged cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Time course of reporter expression during lactation.(a) Expression of mCherry in somatic cells and (b) skimmed milk, collected from colostrum (cd1), and at days 3, 6, 12, 26 and 33 (d3-d33) of lactation; M, size marker; cc, control colostrum from wildtype sow; neg., negative milk control; pos, positive milk control (from mCherry sow). Left immunoblot with anti-mCherry antibody, and right corresponding Coomasssie stained gel (loading control). Note that the colostrum of the transgenic sow (cd1) did not show detectable mCherry expression. In the somatic cells the mCherry signal comes up at d3 (arrow), and in the skimmed milk at day 6 (arrow). The signal intensity is normalized to the marker bands and the positive control, suggesting that the majority of mCherry is deposited in the somatic cells, and released to the skimmed milk by membrane-damaged cells.
Mentions: A time-course experiment to follow the expression of the reporter proteins from day 1 of lactation (colostrum) to weaning is shown in Fig. 5. Interestingly, in colostrum no expression of the reporter (mCherry) could be detected. The mCherry expression started in the somatic cells from day 3 and was then detectable at high levels until weaning. In the skimmed milk, expression of mCherry was first detectable at day 6 and was then continuously present until weaning, but in lower levels than in the somatic cells (Fig. 5). The total amounts of exfoliated cells were similar in the milk from transgenic and control wildtype sows (Supplementary Fig. S2).

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.