Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits

<div><p>Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.</p></div

Macroscopic excitation of the Venus fluorescence protein in tear and oral saliva samples.

<p>(A) Venus-specific fluorescence in tear and (B) saliva samples. -C -negative control; He -Heterozygous; Ho -homozygous. The same parameters were used to detect the fluorophore protein as described in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187214#pone.0187214.g001" target="_blank">Fig 1</a>).</p

Genotypes of the analyzed rabbits.

<p>Genotypes of the analyzed rabbits.</p

Fluorescence of SB-CAG-Venus lactating transgenic rabbit mammary gland.

<p>(A) Tissue section of a heterozygote SB-CAG-Venus transgenic doe showed direct Venus fluorescence at 400X magnification. (B) The section of a non-transgenic doe did not show specific fluorescence under identical conditions. Nuclei were stained with Topro-3-iodide (blue). Scale bars (bottom right) in all images are 50 μm.</p

Western blot analysis of milk fractions.

<p>(A) Milk fractions of a wild-type doe: 1- Whey fraction, (#3014), 2- Fat fraction, (#3014), 3- Milk cell fraction (#3014);Milk fractions of a heterozygous doe: 4- Whey fraction (#4034), 5- Fat fraction (#4034), 6- Milk cell fraction (#4034);Milk fractions of a homozygous doe: 7- Whey fraction, (#4035), 8- Fat fraction, (#4035), 9- Milk cell fraction (#4035), (20 μg/slot). Calculated recombinant protein concentrations are also given in ng/μl.(B)Dilution series of a recombinant GFP-fusion protein (58 kDa).10–250 ng, 11–125 ng, 12–62.5 ng, 13–31.25 ng. (C)Expression of Venus fluorophore in saliva (D) tear fluid (E) seminal plasma samples of the SB-CAG-Venus homozygote, heterozygote and control bucks. 14, 17, 20: #4020 homozygote, 15, 18, 21: #4017 heterozygote, 16, 19, 22: control buck.</p