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Cogburn awarded $1.8 million for gene mapping

NEWARK, DE.--Larry Cogburn, University of Delaware professor of molecular endocrinology, has won a highly competitive $1.8 million grant from the U.S. Department of Agriculture (USDA) Initiative for Future Agriculture and Food Systems for functional mapping of growth regulating genes in broiler chickens.

“We want to identify novel ways to produce lean, healthier chickens because chicken is the principal source of high quality protein in the American diet and that of most people in developed countries,” Cogburn said, noting that despite the importance of chicken in the human diet, research on the chicken genome lags behind that of other animal genomes.

“The Human Genome Project has placed powerful new tools on everybody’s bench top with one notable exception – those of us who work with avian species. The USDA now is placing an emphasis on functional genomics research in chickens,” he said. “We have a lot of catching up to do.”

Cogburn hopes to identify genes important for the regulation of growth and body composition in broiler chickens. Specifically, he is looking for the genes that regulate growth rate, fat deposition and muscle yield, which are unknown at this time.

New era of avian genetics

For this four-year, multi-disciplinary, multi-institutional project, Cogburn has formed a consortium with Tom Porter from the University of Maryland, Sam Aggrey from the University of Georgia and Jean Simon from the Institut National de la Recherche Agronomique, the French equivalent of the USDA.

“Our goal is to identify the critical steps in metabolic and endocrine pathways of broiler chickens so they can be genetically selected to produce elite lines of broiler breeders,” Cogburn said.

Cogburn proposes to identify these genes by continuing the work he began several years ago using French lines of chickens. These are the best genetic models available, he said, and they are used extensively in nutritional and endocrine research.

Assisting with this research at UD is associate professor Lídia Rejtö, who will be developing the bioinformatics programs to analyze and visualize gene expression data.

Cogburn took a sabbatical leave in France in 1996 to begin this work, which is centered on gene expression in two distinct genetic populations of chickens. One population was divergently selected for growth rate – the fast growing versus the slow growing. The other population was divergently selected for fatness versus leanness.

“We mated the fastest growing to the fastest growing, and the slowest growing to the slowest growing, the fattest to the fattest, and the leanest to the leanest. We are looking for the genes responsible for these extremes in production traits, so these French lines are very important genetic models,” he said.

Cogburn collected liver, fat and muscle tissue for this initial test study and plans to return to France shortly to begin another longitudinal study. This time he will collect pituitary/hypothalamus tissue in addition to liver, fat and muscle. Eventually, Cogburn will identify the genes important for the regulation of growth and body composition through the use of microarrays, which are considered the discovery platform for functional genomics.

Microarray technology

According to Cogburn, microarrays allow biologists to quantify the differential expression (high versus low levels) in genes — in this case, in chickens. These snippets of DNA — called cDNA — are immobilized at high density with a gridding robot on 5- by 7-centimeter nylon membranes.

To make the DNA microarray, the messenger RNAs of all genes expressed in tissues are first extracted, a complementary copy (cDNA) of each gene is then made and the cDNA is cloned in bacteria. This is called a cDNA library because each library contains a copy of all of the genes expressed in a particular tissue.

The cDNA library is then submitted to a genomic-scale high throughput sequencing facility, Cogburn said. Finally, the cDNA are printed as thousands of spots on nylon filter or glass slide microarrays.

Cogburn said the chicken DNA microarrays used in his initial studies originally were developed by University of Delaware researchers Joan Burnside and Robin Morgan from an activated immune cell cDNA library. Burnside and Morgan recently received a $950,000 grant from the USDA for the further development of genetic tools.

“We know what the sequence of a gene is before we print them on the filter,” Cogburn said. “That’s the power of microarrays.”

He said software is being developed so that one can point to the location of a gene spotted on a microarray and the gene sequence will be revealed. A link to the public database — called GenBank — will show the sequence in other organisms. “You can even see the list of the papers that have been published on this gene,” he said.

“I’ll be comparing the expression of thousands of genes in the slow and fast growing birds and in the fat and lean birds with the hope of defining critical steps in key regulatory pathways,” Cogburn said. " I’ll have four different tissues, and I’ll have a hundred of these filters for every tissue, and we plan on examining the expression of 5,000 genes for every tissue, so we will have thousands of spots, or expressed genes, to analyze in all.

“I’ve been working 20 years in this field and I’ve only studied two genes,” he said. “Now, with microarray technology, we can examine thousands of genes at a time, and this is just the beginning.”

Gene involved in the genesis of fat identified in chickens

Cogburn said he has found several important differentially expressed genes already but learned firsthand in 1997 what a powerful tool DNA microarrays can be for functional genomics.

“In the very first microarray I did on the liver from French chicken lines, I found the differential expression of a gene called Spot 14,” he said. “This gene is expressed at very high levels in the liver of birds that grow rapidly and accumulate excess fat. In birds that grow slowly and don’t accumulate fat, there is no Spot 14 expression.

“It turns out that Spot 14 controls the synthesis of all six enzymes involved in the synthesis of fat.”

According to Cogburn, Spot 14 is a nuclear transcription factor, called thyroid hormone inducible liver protein. The discovery of this regulatory gene on the microarray was interesting, he said, because it had first been identified in 1984 in rats through a traditional lab method called two-dimensional electrophoresis. This method separates proteins on a gel in two directions, both by electric charge and by molecular weight. Spot 14 marked the position of this protein on the gel. This traditional lab method, however, reveals nothing about the sequence of the gene.

Cogburn pointed out that microarrays alone are not the answer; the appropriate experimental models are needed, too — in this case, the French lines of broiler chickens.

Comparative genomics

There is much human interest in genes that regulate fat synthesis in animals because of a commonality in genomes across species. Cogburn identified Spot 14 in chickens about three years ago. In July, a paper was published on the use of microarrays to demonstrate a 10-fold increase in Spot 14 in the liver tissue of rats. These rats had been fed an active thyroid hormone, as compared with the control rats that were fed a goiter-causing chemical that blocks thyroid hormone synthesis. The identification in Spot 14 both in chickens and rats is an example of this commonality across species.

Cogburn also noted that a hormone, called leptin, recently was identified as the obesity protein in fat mice.

Other research objectives

After Cogburn has printed all his tissue-specific DNA microarrays, he will use them for global gene expression profiling to try to identify genes or clusters of genes responsible for these extremes of growth rate and body composition found in the unique French chickens. Then, he will look for Quantitative Trait Loci (QTLs), which are regions of the chromosome that determine expression of production traits associated with fast growth rates but low body fat content.

This technology may one day provide a more economical alternative to the poultry industry for identifying potential breeders in the embryonic stage. Unlike chickens raised for egg production, which sexually mature in about 13-15 weeks, broilers are slow growing birds. They take 30-32 weeks to reach sexual maturity. Cogburn said the goal is to be able to take blood from the broiler embryo to predict its potential development as a breeder at maturity.

“We should be able to predict the genetic potential of a chick even before it is hatched,” he said. “Once all the genes that contribute to obesity in chickens are identified, poultry breeders can select against those traits, reversing the results.

“Chicken is the most efficient animal besides fish in converting feed energy into animal products, but still chickens accumulate excessive body fat. This means they are less healthy and more prone to metabolic diseases. We want to divert more feed energy into protein, rather than fat, so chickens are healthier themselves, plus leaner and healthier for us to eat.”

When these goals have been met, the final objective of this research will be to locate and map individual genes and QTLs on chicken chromosomes. While humans have 23 sets of chromosomes, chickens have eight large chromosomes and a large number of smaller ones.

Cogburn, who first raised chickens as a 6-year old boy on his father’s farm in the mountains of North Carolina, said microarrays are the key in the identification of genes important for all kinds of production traits in chickens.

“With almost $3 million in funding our department has received from the USDA for chicken genomics, we are extremely lucky,” he said. “We are going to be in a pivotal role in the development of these tools and the discovery of many genes that control growth and development of the broiler chicken.”

Contact: Pat McAdams, (302) 831-1356, mcadams@udel.edu

Nov. 8, 2000