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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.


Concept for high level mammary epithelium-specific expression of recombinant proteins driven by a ubiquitously active promoter.The recombinant protein is separated by a floxed stuffer DNA sequence from the CAGGS promoter. The stuffer DNA contains stop codon sequences and thereby suppresses expression. On a second element a mammary epithelium (ME)-specific promoter drives a Cre cDNA. Only in mammary epithelium cells the Cre cDNA is transcribed, and activates the recombinant protein construct by Cre-mediated deletion of the stuffer. As a result, high level expression is restricted to mammary epithelial cells.
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f6: Concept for high level mammary epithelium-specific expression of recombinant proteins driven by a ubiquitously active promoter.The recombinant protein is separated by a floxed stuffer DNA sequence from the CAGGS promoter. The stuffer DNA contains stop codon sequences and thereby suppresses expression. On a second element a mammary epithelium (ME)-specific promoter drives a Cre cDNA. Only in mammary epithelium cells the Cre cDNA is transcribed, and activates the recombinant protein construct by Cre-mediated deletion of the stuffer. As a result, high level expression is restricted to mammary epithelial cells.

Mentions: For recombinant proteins, such as growth factors, where ectopic expression outside the udder is undesired, the here describe system may be refined by combining it with the Cre recombinase system. A construct with a milk promoter driving the Cre-recombinase and a second construct with a CAGGS promoter, separated by a loxP flanked stop cassette from the gene of interest, would restrict the expression to the mammary gland, but still benefit from the strong transcriptional activity of the CAGGS promoter (Fig. 6). The proposed constructs may be combined on one cassette, or used in a binary approach. Recently, we established a simple and efficient multiplexing approach for transgenesis in cattle68, showing that advanced genome engineering techniques are nowadays sufficiently developed to perform this kind of gene transfer in livestock.


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)

Concept for high level mammary epithelium-specific expression of recombinant proteins driven by a ubiquitously active promoter.The recombinant protein is separated by a floxed stuffer DNA sequence from the CAGGS promoter. The stuffer DNA contains stop codon sequences and thereby suppresses expression. On a second element a mammary epithelium (ME)-specific promoter drives a Cre cDNA. Only in mammary epithelium cells the Cre cDNA is transcribed, and activates the recombinant protein construct by Cre-mediated deletion of the stuffer. As a result, high level expression is restricted to mammary epithelial cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Concept for high level mammary epithelium-specific expression of recombinant proteins driven by a ubiquitously active promoter.The recombinant protein is separated by a floxed stuffer DNA sequence from the CAGGS promoter. The stuffer DNA contains stop codon sequences and thereby suppresses expression. On a second element a mammary epithelium (ME)-specific promoter drives a Cre cDNA. Only in mammary epithelium cells the Cre cDNA is transcribed, and activates the recombinant protein construct by Cre-mediated deletion of the stuffer. As a result, high level expression is restricted to mammary epithelial cells.
Mentions: For recombinant proteins, such as growth factors, where ectopic expression outside the udder is undesired, the here describe system may be refined by combining it with the Cre recombinase system. A construct with a milk promoter driving the Cre-recombinase and a second construct with a CAGGS promoter, separated by a loxP flanked stop cassette from the gene of interest, would restrict the expression to the mammary gland, but still benefit from the strong transcriptional activity of the CAGGS promoter (Fig. 6). The proposed constructs may be combined on one cassette, or used in a binary approach. Recently, we established a simple and efficient multiplexing approach for transgenesis in cattle68, showing that advanced genome engineering techniques are nowadays sufficiently developed to perform this kind of gene transfer in livestock.

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.