The objective of this proposal is to identify genes whose expression is regulated differentially during the blastocyst, egg cylinder, and primitive streak stages of mouse embryogenesis. One commonly used strategy for the identification and subsequent characterization of important gene transcripts is the construction and analyses of cDNA libraries. The principal limitation to this approach in early mammalian embryogenesis is the amount of tissue (and thus RNA) per embryo. We have modified standard procedures for cDNA cloning, and have developed a new procedure using polymerase chain reaction to efficiently clone small amounts of RNA. We have constructed and characterized a large cDNA library from blastocysts which has a proportional representation of RNA species. We are currently generating libraries to inner cell mass, early egg cylinders, and primitive streak embryos. We propose in this application to identify sets of genes that show stage specificity and tissue-specific expression with the ultimate goal of determining how the expression of these sets is regulated. cDNAs will be identified by differential screens, partially sequenced, and homologies sought. In situ hybridization will be used to examine the spatial distribution of gene expression within embryos. Additionally, the libraries will be probed for conserved sequences that appear to regulate transcription during development (such as homeobox, paired box, and """"""""zinc finger"""""""" protein domains), and for oncogenes and growth factors that influence morphogenesis and cell differentiation (TGF-alpha, TGf-beta EGF receptor and insulin-like growth factor). The abundance and tissue distribution of transcripts of these genes will be determined.
| Urven, L E; Weng, D E; Schumaker, A L et al. (1993) Differential gene expression in fetal mouse germ cells. Biol Reprod 48:564-74 |
