The experimental repertoire of the chick embryo includes:

• Surgical manipulations and tissue grafting
• Retrovirus-mediated gene transfer
• Electroporation of developing embryos
• Embryo culture

• Limb development (Fallon, Ide, Izpisua-Belmonte, Martin, Nisswander, Nohno, Tabin, Tickle)
• Neurogenesis (Anderson, Cepko, Le Douarin, Jessell, Lumsden, Tessier-Lavigne)
• Axis development (Le Douarin, Schoenwolf, Stern)
• Somitogenesis (Emerson, Lassar, Le Douarin, Ordahl, Pourquié, Tabin)
• Left-right asymmetry (Noji, Ros, Tabin)
• Integument development (Chuong, Dhouailly, Morgan, Nisswander)

Chicken eggs are plentiful, inexpensive and extraordinarily convenient. Large numbers of eggs can be incubated at one time to obtain embryos at precise stages of development. Chick embryos are much cheaper to maintain and study than mouse embryos. Most institutes' facilities for maintaining and growing transgenic mice are already inadequate, and it is inevitable that this shortage of space will become more severe, even if funding is diverted into expanded infrastructure.

The chick represent the model system which most resembles other higher vertebrates while still permitting experimental intervention in ovo. As such it represents an important complement to mouse model systems.

Chicks offer a further unique system for studying cellular processes: DT40 cells whose high-frequency of mitotic recombination allows the ready generation of somatic cell lines homozygous for targeted mutations. This system is already being used to analyze many gene products that control general processes, such as cell-cycle control, chromosome structure, etc.

Rapid advances are being made in chicken transgenics, ES cell technology and the cryopreservation of sperm, blastdisc cells, primordial germ cells and ES cells. These newer technologies, coupled with the already well established experimental manipulations, will make the chicken an extremely useful and economical organism for studying vertebrate development and for the generation of model systems for human diseases.

Perhaps the major current barrier to efficient use of the above biological systems in the chick is the extensive effort spent re-isolating chick homologues of human, mouse or Drosophila genes. This is usually conducted in laboratories whose major expertise lies elsewhere, in developmental and cellular biology, and so often involves considerable consumption (waste) of time, effort and money. This hurdle could be overcome by a relatively small investment in a chick genome project conducted by professional genomics laboratories.

We propose four approaches to greatly enhance the contributions of the chicken in studies on developmental processes and as model systems for human diseases. They are:

• Generate a chicken EST database with support for data management.
• Generate a physical map of the chicken genome.
• Identify and support relevant mutant chicken repositories.
• Establish a unified website for chicken genomics.

A) Generate a chicken EST database.

A series of normalized cDNA libraries would be used to determine ~200,000 ESTs. The primary need for chick ESTs is to generate probes to analyze endogenous gene expression. Normal gene expression profiles are an important part of interpreting the effects of gain or loss of function experiments. Furthermore, analysis of gene expression has proven to be an effective way to assess the effects of both gene and embryological manipulations. A second need for chick ESTs would be to clone full-length chick sequences to manipulate gene function.

With current technology, the generation of an EST database could be achieved very rapidly and at an estimated cost of $1-2 million. The savings in effort and resources currently spent on gene isolation alone would justify the latter expenditure. Individual cDNA libraries would be derived from a series of hand-dissected tissue samples which, together, would enlarge the diversity of low level, tissue specific transcripts represented in the database. These would derive from a genetically defined chick strain, e.g. that for which ES cells are available. Tissues for the libraries would be isolated by various laboratories within the chick community according to their biological speciality and suitable normalization would minimize library redundancy.

In the slightly longer-term, the ESTs would be used to generate gridded cDNA microarrays for expression screening. The chick will offer a unique opportunity for in vivo expression screening in developmental and pathological contexts.

B) Generate a physical map of the chicken genome.

Techniques are currently being developed for analyzing gene regulation in chick embryos. These include electroporation of embryos, retroviral vectors for analyzing gene function and regulation, gene knock-out utilizing ribozymes, and chicken ES cells for germ-line studies. These experimental approaches are much cheaper and faster than their mouse equivalents which, as mentioned above, require facilities that are, or soon will be, saturated in most institutions. The chick also offers excellent material for analyzing gene regulatory sequences and genome organization. The chick genome is one-third the size of that of the mouse. In addition, it has been argued that most chick genes are organized in gene-rich microchromosomes at densities equivalent to those of Fugu. Analysis of gene organization in the chick would also contribute to our understanding of human gene and chromosome organisation as syteny between chick and human chromosomes seems to be much higher than that between mice and humans.

These experimental advantages that are offered by the chick need to be supported by a physical map of the chick genome that would allow efficient recovery of chick genes. There are a number of laboratories associated with chicken genomics. These are listed in Burt and Cheng (See chicken matrix for reference) and any efforts that result from the present proposal should be integrated with existing efforts.

A library of BAC clones sufficient to provide 10X genome coverage would be constructed and gridded. This would be supplemented by a 3X YAC library. These would be derived from the same chick strain as used in the EST project. Filters of gridded clones would be made available to investigators who would screen and identify specific genes. Such gene identification would also aid in clone assembly. Assembly of the physical map would involve fingerprinting techniques utilizing robotic sequencing techniques. Clone end-sequences would also be determined. Assembly software and other informatics could be adapted from already existing software developed for other genome projects. It is anticipated that complete assembly of the physical map could be largely completed within 3 years.

Although complete genome sequencing is not proposed at this time, this should ultimately be considered to maximize the usefulness of this organism in studying development and generating model systems for human diseases.

C) Identify and support relevant mutant chicken repositories.

A number of chick mutants have played key roles towards our understanding of morphogenesis in vertebrates. This is particularly true for the development of the limb, the integument and the skeleton. In addition the chicken has proven to be useful as model systems for autoimmune forms of avian vitiligo, scleroderma, thyroiditis, scoliosis and the autosomal recessive form of muscular dystrophy. Thus in addition to their enormous value in studies on development these mutants are also useful as models for human diseases. A listing of all chicken mutations has been compiled as part of a report entitled "Avian Genetic Resources at Risk: An Assessment and Proposal for Conservation of Genetic Socks in the USA and Canada". The complete data on the extant genetic resources of the chicken are presented in Appendix 2 of the report which will be published by the Genetic Resource Conservation Program of the University of California at Davis.

The mutations that are relevant to the mission of the NIH should be identified and supported.

D) Establish a unified website for chicken genmomics.

A number of websites on chicken genomics already exist. These are:

http://www.ri.bbsrc.ac.uk/chickmap

http://poultry.mph.msu.edu/mapsnew.html

http://ag.ansc.purdue.edu/poultry/

http://www.zod.wau.nl/vf/chickensite/chicken.html

http://tetra.gig.usda.gov:8400/chickgbase/manager.html

It is proposed that the information on chicken genomics generated from proposals (A) and (B) above be integrated with existing public information resources. In this manner all chicken genomic information would be made available to the scientific community. Such a resource would maximize the usefulness of the chicken in studying development and human diseases.

SUMMARY CONCLUSIONS

• The chick is the cheapest advanced model system for studying embryonic development and gene function in higher vertebrates.
• It offers several unique experimental advantages, most particularly, accessibility and manipulatibility.
• The above proposal would greatly extend the contributions that can be made by the chicken in fundamental biological research.
• The above projects would be cheap and rapid, provide excellent value for money, and be of immediate utility to a wide community.