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

Augmented humoral immune response in transgenic rabbits.

<p><b>A–B</b>. Tg+/− and wt rabbits (4 and 8 animals, respectively) were immunized with OVA. After the booster immunization the OVA-specific IgG titers were nearly double in Tg rabbits as compared with the wt animals. We found that the total IgG levels rose steadily after immunization and reached peak levels on day 49 in Tg and wt animals. <b>C–D</b>. Tg+/− and wt rabbits (5 and 5, respectively) were immunized with TNP-BSA. The mean TNP-specific IgG level was higher in Tg rabbits as compared to their wt controls at the peak of the immune response, on day 49, one week after the second booster immunization with an almost doubled level of total IgG as compared to their wt controls. <b>E–F</b>. The same rabbits which had been immunized with TNP-BSA, were immunized with a conserved influenza hemagglutinin epitope (HA2) conjugated to KLH. The mean of the HA2-specific IgG levels was double at the peak of the immune response, on day 56, two weeks after the second booster immunization, with an almost doubled level of total IgG as compared to their wt controls. Values shown are the means ± SEM. (*, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.).</p

Cytoplasmic domains of the FcRn sequences most likely reflect their phylogenetic position.

<p>Marsupials (possum, opossum, and wallaby) have relatively short cytoplasmic domains composed of 27–28 amino acid residues. The next phylogenetic step resulted in clades Atlantogenata and Boreoeutheria. The only (predicted) sequence we found belonging to Atlantogenata (elephant) shows a 7–8 amino acid longer cytoplasmic domain as compared to Marsupials. Boreoeutheria is composed of the sister taxa Laurasiatheria and Euarchontoglires. Species belong to Euarchontoglires analyzed so far (human, chimp, gorilla, orangutan, gibbon, rhesus, marmoset, lemur, rabbit, pika, squirrel, hamster, rat and mouse) preserved these extra amino acids of the FcRn C-terminal with the exception of the guinea pig (based on its predicted amino acid sequence). Rabbit lost five amino acids in a more N-terminal (or middle) part of the cytoplasmic domain. As pika (Ochotona), another Lagomorphs, possesses these residues, the five amino acid deletion is thus specific of either rabbit or Leporidae family. Animals belong to the Laurasiatheria clade (bovine, sheep, pig, horse, bat, dog and panda) lost 10 amino acids of their FcRn C-terminals. Phylogenetic tree was created based on Prasad et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028869#pone.0028869-Prasad1" target="_blank">[45]</a> where some branch lengths were optimized for clarity and space.</p

Rabbit embryos start to express FcRn by 6 dpc, close to the implantation time.

<p>Rabbit FcRn α-chain expression was analysed in rabbit blastocysts and embryos at different time points by PCR. L-ladder, 1–3.5 dpc rabbit blastocyst, 2–4.5 dpc rabbit blastocyst, 3–6 dpc rabbit embryo, 4–9 dpc rabbit embryo, 5–13.5 dpc rabbit embryo, 6 - negative control (DNA omitted).</p

Reduced IgG catabolism in Tg rabbits that carry two extra copies of the rabbit FcRn.

<p>We have analyzed the half-life of rabbit IgG in Tg (homozygous; +/+) and wt rabbits from days 2–13 after injecting OVA-specific rabbit IgG i.v. into these animals. <b>A–B.</b> Our analysis showed that the Tg rabbits have increased serum persistence of rabbit IgG as the beta phase half-lives of the IgG were 7.1±0.5 days (mean ± SEM) as compared to their controls which showed 5.3±0.3 days. <b>C.</b> This difference may be even greater as non-immunized Tg rabbits have higher total IgG levels as compared to their controls. Values shown are the mean ± SEM. (*, <i>P</i><0.05). The experiment was repeated two times with similar results.</p

Detection of the rabbit FcRn in Western blot.

<p>This Western blot shows that the chicken antibody strongly and specifically reacted with the soluble recombinant bovine FcRn which was used for immunization (its estimated molecular weight is 30 kDa), a ∼40-kDa band that is consistent with the known molecular weight of the bovine FcRn α-chain in the protein extract from a bovine FcRn stably transfected cell line (B4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028869#pone.0028869-Kacskovics4" target="_blank">[46]</a> which strongly expresses the functional form of the bovine FcRn and a ∼40-kDa band that is consistent with the calculated molecular weight of the rabbit FcRn α-chain in the protein extract from the rabbit yolk sac from 24 dpc fetuses.</p