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.


Related in: MedlinePlus

High level expression of Venus in transgenic milk.(a) Sedimented milk cells (top) and skimmed milk (bottom) from a Venus transgenic sow shown under specific excitation of Venus (50 ms exposure). Arrow points to pellet of milk cells at the bottom of a 1.5 ml centrifugation tube (isolated from 1 ml of milk). (b) Corresponding brightfield illumination. (c) Wildtype milk cells (top) and skimmed milk fraction shown under specific Venus illumination. (d) Same samples as in (c) shown under brightfield illumination. (e) Milk cells (top, arrow) and fat fraction (bottom) of milk from a Venus transgenic sow shown under specific excitation of Venus. Note that the fat fraction displays a reduced fluorescence relative to the cell pellet. Arrow points to cell pellet. (f) Same samples as in (e) shown under brightfield illumination. (g) Top: Venus immunoblot of fractions elution from a Sephadex G50 column loaded with Venus containing skimmed milk. M, size marker (Magic Mark); F0 before loading on column; F4-F11, collected fractions of flow-through. Arrow indicates Venus protein, migrating at an apparent molecular weight of 29 kD. Bottom: corresponding Coomassie-stained gel. (h) Immunoblots of Venus (top) and tubulin (bottom) from milk cells (c1, c2) and skimmed milk (w1, w2) fractions of two different samples of Venus-containing milk, and from milk of a wildtype sow (wt-c, wt-w); +, positive control of purified Venus; M, size marker (Magic Mark).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: High level expression of Venus in transgenic milk.(a) Sedimented milk cells (top) and skimmed milk (bottom) from a Venus transgenic sow shown under specific excitation of Venus (50 ms exposure). Arrow points to pellet of milk cells at the bottom of a 1.5 ml centrifugation tube (isolated from 1 ml of milk). (b) Corresponding brightfield illumination. (c) Wildtype milk cells (top) and skimmed milk fraction shown under specific Venus illumination. (d) Same samples as in (c) shown under brightfield illumination. (e) Milk cells (top, arrow) and fat fraction (bottom) of milk from a Venus transgenic sow shown under specific excitation of Venus. Note that the fat fraction displays a reduced fluorescence relative to the cell pellet. Arrow points to cell pellet. (f) Same samples as in (e) shown under brightfield illumination. (g) Top: Venus immunoblot of fractions elution from a Sephadex G50 column loaded with Venus containing skimmed milk. M, size marker (Magic Mark); F0 before loading on column; F4-F11, collected fractions of flow-through. Arrow indicates Venus protein, migrating at an apparent molecular weight of 29 kD. Bottom: corresponding Coomassie-stained gel. (h) Immunoblots of Venus (top) and tubulin (bottom) from milk cells (c1, c2) and skimmed milk (w1, w2) fractions of two different samples of Venus-containing milk, and from milk of a wildtype sow (wt-c, wt-w); +, positive control of purified Venus; M, size marker (Magic Mark).

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)

High level expression of Venus in transgenic milk.(a) Sedimented milk cells (top) and skimmed milk (bottom) from a Venus transgenic sow shown under specific excitation of Venus (50 ms exposure). Arrow points to pellet of milk cells at the bottom of a 1.5 ml centrifugation tube (isolated from 1 ml of milk). (b) Corresponding brightfield illumination. (c) Wildtype milk cells (top) and skimmed milk fraction shown under specific Venus illumination. (d) Same samples as in (c) shown under brightfield illumination. (e) Milk cells (top, arrow) and fat fraction (bottom) of milk from a Venus transgenic sow shown under specific excitation of Venus. Note that the fat fraction displays a reduced fluorescence relative to the cell pellet. Arrow points to cell pellet. (f) Same samples as in (e) shown under brightfield illumination. (g) Top: Venus immunoblot of fractions elution from a Sephadex G50 column loaded with Venus containing skimmed milk. M, size marker (Magic Mark); F0 before loading on column; F4-F11, collected fractions of flow-through. Arrow indicates Venus protein, migrating at an apparent molecular weight of 29 kD. Bottom: corresponding Coomassie-stained gel. (h) Immunoblots of Venus (top) and tubulin (bottom) from milk cells (c1, c2) and skimmed milk (w1, w2) fractions of two different samples of Venus-containing milk, and from milk of a wildtype sow (wt-c, wt-w); +, positive control of purified Venus; M, size marker (Magic Mark).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: High level expression of Venus in transgenic milk.(a) Sedimented milk cells (top) and skimmed milk (bottom) from a Venus transgenic sow shown under specific excitation of Venus (50 ms exposure). Arrow points to pellet of milk cells at the bottom of a 1.5 ml centrifugation tube (isolated from 1 ml of milk). (b) Corresponding brightfield illumination. (c) Wildtype milk cells (top) and skimmed milk fraction shown under specific Venus illumination. (d) Same samples as in (c) shown under brightfield illumination. (e) Milk cells (top, arrow) and fat fraction (bottom) of milk from a Venus transgenic sow shown under specific excitation of Venus. Note that the fat fraction displays a reduced fluorescence relative to the cell pellet. Arrow points to cell pellet. (f) Same samples as in (e) shown under brightfield illumination. (g) Top: Venus immunoblot of fractions elution from a Sephadex G50 column loaded with Venus containing skimmed milk. M, size marker (Magic Mark); F0 before loading on column; F4-F11, collected fractions of flow-through. Arrow indicates Venus protein, migrating at an apparent molecular weight of 29 kD. Bottom: corresponding Coomassie-stained gel. (h) Immunoblots of Venus (top) and tubulin (bottom) from milk cells (c1, c2) and skimmed milk (w1, w2) fractions of two different samples of Venus-containing milk, and from milk of a wildtype sow (wt-c, wt-w); +, positive control of purified Venus; M, size marker (Magic Mark).
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.


Related in: MedlinePlus