Competitive bioreactor hens on the horizon.

The hen has long held promise as a low-cost, high-yield bioreactor for the production of human biopharmaceuticals in egg whites using genetic engineering. Two separate reports have recently appeared indicating the production of substantial levels of human monoclonal and single chain antibodies (>3 mg and >150 mg, respectively) in eggs of transgenic hens. These promising findings indicate that the hen is close to becoming a competitive manufacturing platform for the production of human biopharmaceuticals.

The repetitive landscape of the chicken genome.

Cot-based cloning and sequencing (CBCS) is a powerful tool for isolating and characterizing the various repetitive components of any genome, combining the established principles of DNA reassociation kinetics with high-throughput sequencing. CBCS was used to generate sequence libraries representing the high, middle, and low-copy fractions of the chicken genome. Sequencing high-copy DNA of chicken to about 2.7 x coverage of its estimated sequence complexity led to the initial identification of several new repeat families, which were then used for a survey of the newly released first draft of the complete chicken genome. The analysis provided insight into the diversity and biology of known repeat structures such as CR1 and CNM, for which only limited sequence data had previously been available. Cot sequence data also resulted in the identification of four novel repeats (Birddawg, Hitchcock, Kronos, and Soprano), two new subfamilies of CR1 repeats, and many elements absent from the chicken genome assembly. Multiple autonomous elements were found for a novel Mariner-like transposon, Galluhop, in addition to nonautonomous deletion derivatives. Phylogenetic analysis of the high-copy repeats CR1, Galluhop, and Birddawg provided insight into two distinct genome dispersion strategies. This study also exemplifies the power of the CBCS method to create representative databases for the repetitive fractions of genomes for which only limited sequence data is available.

Rapid and improved method for windowing eggs accessing the stage X chicken embryo.

The chick stage X blastoderm is routinely accessed through a small window in a freshly laid egg. However, windowing severely compromises embryo survival with hatch rates as low as a few percent. We previously reported a simple modification to the standard method that reduced introduction of air into the sealed egg and improved the hatchability to 32%. Here, we describe an even simpler and more rapid method for sealing a windowed egg using hot glue or paraffin in which the hatch rate increased to an average of 63% of the unwindowed control eggs. The primary reason for low hatchability can be attributed to air trapped within the egg during windowing and/or leakage during incubation, as shown by increased lethality by artificially introducing air into windowed and sealed eggs. Although the hatch rate was considerably improved, air can still enter the egg during incubation and is likely to account for less than 100% hatchability of the sealed eggs. The success of this new windowing method will facilitate high throughput for the production of transgenic birds and find use in developmental biology, toxicity testing, and avian disease research.

Biologically active human interferon alpha-2b produced in the egg white of transgenic hens.

We have previously described the expression of a bacterial protein in the egg white of transgenic chickens using a replication-deficient retroviral vector. Here we report the expression of a glycosylated human protein, interferon alpha-2b (hIFN), in the egg white of transgenic hens. The hIFN secreted into the egg white was biologically active as determined by a viral inhibition assay. Purification and carbohydrate analysis of the hIFN expressed in egg white revealed that two of the six major glycosylated hIFN species match the naturally occurring human hIFN glycovariants. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active, and glycosylated proteins in egg white.

Avian transgenesis: progress towards the promise.

The hen has long held promise as a low cost, high-yield bioreactor for the production of human biopharmaceuticals in egg whites. A typical egg white contains 3.5-4.0 grams of protein, more than half of which comes from a single gene (ovalbumin). Harnessing the power of the gene to express a recombinant protein could yield up to a gram or more of the protein in the naturally sterile egg. Accordingly, a major effort has been underway for more than a decade to develop robust methods for modification of the chicken genome. This effort intensified in the mid-1990s when several avian transgenic companies entered the scene. Progress has been made in that time but much remains to be done.

Expression of exogenous protein in the egg white of transgenic chickens.

Using a replication-deficient retroviral vector based on the avian leukosis virus (ALV), we inserted into the chicken genome a transgene encoding a secreted protein, beta-lactamase, under the control of the ubiquitous cytomegalovirus (CMV) promoter. Biologically active beta-lactamase was secreted into the serum and egg white of four generations of transgenic chickens. The expression levels were similar in successive generations, and expression levels in the magnum of the oviduct were constant over at least 16 months in transgenic hens, indicating that the transgene was stable and not subject to silencing. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active proteins in egg white.