Modern techniques in Molecular Biology including cDNA arrays and automated sequencing have made possible the identification of genes expressed by a particular type of cell. The most common strategy to identify novel cell-type specific genes is to sequence cDNA clones obtained from the reverse transcription of mRNA using oligo dT and reverse transcriptase (RT). Due to limitations of the technique, these cDNAs often consist of partial clones of the 3' region of the mRNA. Since this 3' region often consists of non-coding sequences, insights into the function of the protein encoded by the mRNA are not possible. Techniques such as """"""""random priming"""""""", optimization of mRNA preparation and improvement of the enzymatic reactions used in cDNA synthesis can increase the yield of full-length product. Even these optimized procedures result in many truncated cDNAs. Furthermore, these procedures require large amounts of mRNA starting material. This laboratory is interested in the developmental regulation of stem cells, a rare population of cells that play a critical role in organogenesis. Genes expressed by these cells are among the most likely to escape identification in the EST database efforts. In this proposal, we propose to develop novel in vitro and in vivo methods to make cDNA from such cell populations.
The Specific aims of this project follow:
Specific Aim No. 1. To make long cDNA libraries from rare cell populations.
Specific Aim No. 2. To develop a method to use random priming to clone the 5' end of the mRNA obtained from rare cells.
Specific Aim No. 3. To develop a method to make cDNA in vivo.
