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Songbird Neurogenomics

Songbird cDNA Transcriptome Database
cDNA microarrays measure the expression of large numbers of genes simultaneously. We are identifying genes active in the songbird brain and creating microarrays with these genes to investigate the molecular basis of learned vocal communication.

Songbird Gene Chip Project

Microarray

We are creating a songbird (zebra finch) brain cDNA microarray gene chip that will contain brain cDNAs from birds undergoing vocal learning and vocal communication. The project involves collaboration with the RIKEN Genomic Sciences Center in Japan, Duke University Center for Genome Technology and Duke Bioinformatics Shared Resource. The Genome Center has a cDNA microarrayer capable of arraying 10,000 cDNAs per slide, robotic colony picker, robotic DNA isolation workstation, and robotic high-throughput DNA sequence analyzer. The Bioinformatic Resource has training courses, high throughput computers, and bioinformatic software. Also see the Songbird Neurogenomics Initiative by another group.

We want to characterize the molecular basis of the songbird vocal communication system. Not enough songbird genes have been previously cataloged to make cDNA microarrays. Therefore the consortium of investigators from Duke University and RIKEN Institute was formed with the following aims:

  1. clone full length cDNAs from a normalized zebra finch brain library that represents mRNAs expressed during commonly studied vocal communication states,
  2. characterize these cDNAs by sequencing, and
  3. use them to generate non-redundant microarrays for asking biological questions.

1. A normalized cDNA library is preferred because it contains more equal representation of genes, which reduces labor and cost need to obtain many unique cDNAs. Full-length cDNAs are needed for translating and transcribing them into proteins in experiments designed to determine gene function. The RIKEN genomics group is experienced with the generation of full-length and normalized cDNAs from their work with the mouse genome.

Using RIKEN's most recent technology, we are creating gene libraries from the brains of adult male zebra finches who were 1. singing undirected song, 2. singing directed song to a female, 3. silent. The purpose is to maximize the differences in the bird's behaviors such that differentially expressed genes will be present in the different brain libraries. The brain mRNA is isolated, converted to cDNAs, the cDNAs then ligated into a viral vector, followed by in-vitro plasmid excision to form pFLC-I songbird containing clones. for this project we expect to find approximately 10,000 unique cDNA clones.

2. The sequences we obtain are run through the DNAprocess software produced at the Duke Bioinformatics Shared Resource, and the annotated results are deposited in our songbird cDNA database. The software processes and organizes the sequences by automatically performing the following functions:

  1. performs base calls for the sequence and checks for sequence quality using phred;
  2. if good quality, finds and clips off vector and primer sequences using cross-match;
  3. takes the remaining cDNA sequences and performs a series of searches that include a Genbank blast search to find identities;
  4. a Gene Ontology database search to infer gene function and assign standard vocabulary;
  5. a UniGene database search to find known tissues of expression and other information;
  6. deposits the annotated sequence into our songbird cDNA database.
cDNA generation process

Process of Generating cDNA clones.
Click to Enlarge.

3. The RIKEN microarray facility, well-known for their production of the mouse 20K microarray set, is being used to generate the initial songbird microarray set. The cDNA molecules on glass slides are called probes, and the labeled cDNA population used to hybridize them are called targets. To construct the probes for the microarrays, isolated plasmid DNA of the selected unique cDNA clones is used to synthesize PCR products of the inserts in 384-well PCR formats. We are also testing using single-stranded antisense RNA strands to improve detection of gene expression from small amounts of tissue.

In the future, as a general service to the songbird and avian neuroscience community, songbird microarray slides will be generated at the Neurosciences Microarray Center at Duke University with our clones and provided to other investigators. The same will be done with the clones themselves.

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