Oviduct-specific expression of two therapeutic proteins in transgenic hens

Abstract

Recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. Ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. Here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to direct synthesis of associated therapeutic proteins to the oviduct. We report the generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in a tightly regulated tissue-specific manner, without any evidence of transgene silencing after germ-line transmission.

Fig. 1.

Schematic representation of the structure of the vectors described after chromosomal integration. Relative positions of lentiviral elements (light gray) and ovalbumin regulatory sequences (dark gray) are indicated, including the EIAV LTRs and packaging signal (Ψ) and the ovalbumin gene ERE; steroid-dependent regulatory element (SDRE), negative regulatory element (NRE), exon 1, intron 1, and the 5′ segment of exon 2 of the ovalbumin transcribed sequence; and the Kozak sequence and coding sequence of either miR24 or hIFNβ1a (CDS). The positions of restriction sites SpeI (S), NcoI (Nc), HindIII (H), and NdeI (Nd) used in restriction analysis are marked. The U3 region of the 3′ LTR is modified such that the integrated provirus lacks an active promoter within the LTR. Relative positions of regulatory elements in the chicken ovalbumin locus are shown.

Fig. 2.

Southern transfer analysis of genomic DNA from individual G₁ birds. (A and B) Samples from 10 birds carrying the OVA-miR24 provirus (A) were digested with HindIII (Ai) or SpeI (Aii) to generate either a junction fragment or a fragment spanning the ovalbumin regulatory sequence, respectively. Samples from two birds carrying the OVA-IFN provirus and 12 birds carrying the EREOVA-IFN provirus (B) were digested with NcoI (Bi) or NcoI/NdeI (Bii) to generate either a junction fragment or a fragment spanning the ovalbumin regulatory sequence, respectively. The G₀ sires of the G₁-IFN birds analyzed are indicated below.

Fig. 3.

Northern blot analysis of total RNA from individual G₁ and G₂ hens. (A–D) Total RNA was extracted from the magnum portion of the oviduct (M), spleen (S), pancreas (P), brain (B), intestine (I), liver (L), and breast muscle (Br) from: two G₁ hens (OR2-4:447 and OR2-4:476, A) and two G₂ hens (OR2-4:25:59 and OR2-4:25:171, B) carrying the OVA-miR24 vector; two G₂ hens carrying the EREOVA-IFN vector (EOI3-13:148:14 and EOI3-13:148:40, C), and a nontransgenic control hen (D). Blots were sequentially hybridized with probes for either miR24 (R) or hIFNβ1a (I), followed by an ovalbumin probe (O) and then 18S ribosomal RNA (18S), with the exception of the control, which was hybridized sequentially with probes for miR24, hIFNβ1a, ovalbumin, and 18S ribosomal RNA.

Fig. 4.

Immunohistochemical detection of miR24 in oviduct sections. (A and B) Sections of the magnum portion of the oviduct of hen OR2-4:25:59 (A) and a nontransgenic control (B) were stained with the nuclear stain Hoescht to visualize individual cells or FITC-anti-human IgG to visualize location of miR24. Tubular gland cells expressing the transgene appear green, whereas epithelial cells lining the tubular glands (indicated by arrows) do not produce miR24.

Fig. 5.

Concentration of recombinant protein in egg white from transgenic hens. (A and B) Egg-white solution was assayed by ELISA for the presence of recombinant miR24 (A) or hIFNβ1a (B). Columns represent the mean concentration of recombinant protein (±SD) in egg white from each of the first 20 eggs and then each subsequent 10th egg up to a maximum of the 150th egg from the hens indicated. G₁ and G₂ hens are indicated above, with individual bird numbers below.

Fig. 6.

Biological activity of recombinant hIFNβ1a in egg white from transgenic hens. Egg white from hens OI2-4:380, EOI2-6:328, and EOI3-13:813 was analyzed in a cytopathic effect assay for antiviral activity. By applying a nonlinear regression (curve fit), the dilution of each sample, which resulted in 50% of cells being protected from Semliki forest virus (EC₅₀), was calculated and compared with the EC₅₀ for reference IFNβ1a to determine hIFNβ1a concentration (activity) in the egg white samples. Estimates of hIFNβ1a measured by ELISA in the same egg white samples are provided for comparison.