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The incredible, edible, and therapeutic egg

James N. Petitte, and Paul E. Mozdziak

doi:10.1073/pnas.0611652104published online Feb 1, 2007;PNAS

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The incredible, edible, and therapeutic eggJames N. Petitte* and Paul E. MozdziakDepartment of Poultry Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC 27695-7608

T

he domestic fowl has a long andunique history, serving multiplepurposes in society and science.

A cursory review of the NobelPrize awards in physiology or medicinesince 1901 points to the significance ofbirds, and the chicken in particular, as thepremier nonmammalian vertebrate animalmodel (see www.fbresearch.org/education/nobels.htm and http://nobelprize.org/nobel

prizes/medicine/laureates). The do-

mestic fowl aided in the discovery ofessential vitamins and gave the first clueto differences between T and B cells. Infact, B cell nomenclature is based on theorigin of B cells from the avian bursa ofFabricius. In addition, the chicken modelprovided the foundation for understand-

ing the chemical processes for vision, in-sights into animal behavior, and our firstintroduction to tumor viruses [e.g., Roussarcoma virus (RSV)] and the cellularorigin of retroviral oncogenes. Eventoday, avian oncogenic viruses provide

valuable models for human disease. Fur-thermore, for many developmental bi-ologists, the avian embryo remains thepremier animal model (1). On the practi-cal side, the general public is protectedfrom yearly influenza outbreaks through

vaccine production in chicken eggs. Inaddition to its scientific and biomedicalimportance, poultry as an agriculturalcommodity has grown over the last 60

years into a global industry providing bil-lions of people with inexpensive high-quality animal protein in the form ofmeat and eggs. Much of the success ofthe poultry industry is directly related tothe application of population genetics forthe selection of commercial lines for effi-cient protein production (2). Today, esti-mates of the cost of egg production in theU.S. hover around 5 cents per egg. Giventhat the albumin from a single egg con-tains 3.6 g of protein, the domesticlaying hen is a very efficient protein biore-

actor. Now, with the report of Lillico et al.(3) in this issue of PNAS, the domesticchicken is poised to become the animalbioreactor for the production of commer-cial quantities of therapeutic proteins ineggs, moving the domestic fowl into therealm of protein bioprocessing.

Generating transgenic chickens hasnot been a quick and easy goal. Sincethe emergence of recombinant DNAtechnology 25 years ago, those who

work with poultry have recognized theadvantage of the avian system as an effi-cient means of producing medically rele-

vant proteins. The avian egg contains

large amounts of protein, and over halfof the protein in egg white or albuminis composed of a single species, ovalbu-

min. Unfortunately, the advantages thatmake the avian system useful in disci-plines such as developmental biologyhave made the task more difficult formanipulating the avian genome. Scien-tists who considered the chicken theirmodel organism of choice watched withsome envy while methods to manipulatethe mammalian genome became increas-ingly more sophisticated, and reportsappeared describing therapeutic proteinproduction in the milk of sheep, goats,cattle, and rabbits (4, 5).

The avian egg, with its large yolk, is

not as transparent as mammalian ova,making it a challenge to view for injec-tion and manipulation. After ovulation,fertilization occurs in the upper repro-ductive tract. Then the egg spends

2426 h traversing the oviduct of thehen to acquire the albumin, shell mem-branes, and finally the eggshell. Duringegg formation, the embryo continues todevelop, and by the time the egg is laid,the embryo can be composed of asmany as 50,000 cells. Once the egg islaid, the self-contained embryo cannotsurvive manipulation without the devel-opment of specific procedures to culturethe nascent embryo through hatching.

The idea of using the domestic fowlfor the production of therapeutic pro-teins has always centered on methodsfor making transgenic poultry. Effortsfor manipulating the avian genome havefocused on three basic approaches: (i)direct DNA injection into the develop-ing zygote, (ii) the culture of avian em-bryonic stem cells or primordial germcells, and (iii) viral vectors to deliverDNA. All three methods have beenused to produce transgenic birds, but

viral vectors are currently the most suc-cessful method (6).

Direct DNA injection was investigatedby Helen Sangs group (7), the first toproduce transgenic chickens through the

injection of DNA into the vicinity of thepronuclei of the newly fertilized zygote.This process required surgical harvesting

of the ovum from the hen at a precisetime after ovulation and the develop-ment of a complex three-stage culturesystem to nurture the embryo throughhatching (8). The efficiency of this pro-cess was low, and only a few transgenicbirds have been produced through DNAinjection.

From the beginning, retroviral vectorswere recognized as a way to producetransgenic birds (6). Viral stocks could beinjected into the egg through a simple

window in the eggshell (Fig. 1). Some ofthe first vectors tested to produce trans-genic chicks were replication-competentRSV and avian leukosis virus (ALV) (9,10). Subsequently, because of the possiblepathogenicity associated with replication-competent viral vectors, reticuloendotheli-osis virus and ALV replication-defective

vectors became the method of choice forthe production of transgenic chickens (11,12). However, early concern over possiblerecombination with endogenous retroviralsequences dampened efforts to use viral

vectors until the last 5 years. Recently,hens were generated that expressedreporter genes such as LacZ and-lactamase (1315). Interestingly, theubiquitous expression of-lactamase re-sulted in some secretion of the proteininto the egg white. This tantalizing result

was later applied to the expression of hu-man IFN--2b in egg white (16). How-ever, germ-line transmission was notoptimal, making it necessary to screenhundreds to thousands of offspring to finda few transgenic birds (Fig. 1). If this inef-ficiency is coupled with the phenomenonof transgene silencing (17, 18), the pro-duction of useful lines of transgenic chick-ens appears risky.

The group at the Roslin Institute (3)addressed the issue of germ-line effi-

ciency and gene silencing through theuse of self-inactivating pseudotypedlentiviral vectors based on the equineinfectious anemia virus (19). Unlike ret-roviral vectors, lentiviruses do notrequire cell division for efficient integra-tion into the host genome, resulting in

Author contributions: J.N.P. and P.E.M. wrote the paper.

The authors declare no conflict of interest.

See companion article on page 1771.

*To whom correspondence should be addressed. E-mail:

jpetitte@ncsu.edu.

2007 by The National Academy of Sciences of the USA

The domestic

laying hen is a very

efficient protein

bioreactor.

www.pnas.orgcgidoi10.1073pnas.0611652104 PNAS February 6, 2007 vol. 104 no. 6 17391740

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founder birds transmitting the vector toas much as 45% of their offspring. Inaddition, the effect of gene silencingappeared minimal. The report of Lillico et al. (3) represents a collaboration be-

tween the Roslin Institute and two com-panies, Oxford Biomedica and Viragen(Scotland). Their work yielded a cap-stone publication in the efforts to de-

velop an efficient system of makingtransgenic birds that deposit high levelsof commercially relevant proteins ineggs using a lentiviral vector. Because ofpackaging-size limitations, a scaled-

down version of the well characterizedovalbumin promoter was constructed todrive the expression of human IFN--1aand a humanized single-chain FvFc

antibody with the potential for treatingmalignant melanoma. In the end, theresulting transgenic birds depositedcommercially significant amounts of atherapeutic antibody and a therapeuticprotein specifically in the egg white.Furthermore, the recombinant IFN wasfunctional in a viral inhibition assay sim-ply by using raw diluted egg white.

Viral vectors are not perfect. Packag-ing size is limited to 10 kb of DNA,so the expression construct must bestreamlined. High viral titers are neces-sary to ensure reasonable rates of germ-line transmission, and viral insertionscan occur anywhere throughout the ge-nome. Recently, additional nonviraltools have been developed to completethe repertoire of choices for taking ad-

vantage of the efficient protein produc-tion of the contemporary laying hen.These methods include the developmentof pluripotent chicken embryonic stemcells derived from the early blastoderm(2022) and the long-term culture ofchicken primordial germ cells, whichhave been used to develop transgenicchickens without the use of viral vectors(23). Unlike the state of the art 20 yearsago, all of the tools, including a freshassembly of the chicken genome, appearto be in place to take advantage of thepotential applications of transgenicchickens.

No one can really predict the futurewhen it comes to the commercializationof science, but in the next 25 years, it isreasonable to expect that, in basic sci-ence, agriculture, and now protein bio-processing, the chicken will continue toprovide benefits to society. In the end,chicken could be the source of severaltherapeutic proteins that are good for

what ails you.

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Fig. 1. A schematic outline of the steps to generate transgenic chickens using viral vectors. (A) Suitable

viral expression constructs are developed and transfected into the appropriate packing cell line. ( B)

High-titer virus is injected beneath the blastoderm of the unincubated egg. ( C) Injected embryos are

culturedfor21 days through hatching.(D) Founder (G0) chicksare raisedto sexualmaturity. (E) Founder

roosters positive for the transgene in semen are mated with wild-type hens, and the G 1 offspring are

screened for the presence of the transgene (illustration by Jennifer Petitte).

1740 www.pnas.orgcgidoi10.1073pnas.0611652104 Petitte and Mozdziak