IN-SITU HYBRIDIZATION AND OLIGOMER PROBES - EVALUATION OF GENE-EXPRESSION DURING DEVELOPMENT

Alteration of gene expression can result in numerous pathologies, incl uding proliferative diseases, functional deficits, and developmental d efects. Recent studies have suggested that changes in the expression d omains of Homeotic (Hox) genes during development are associated with the induction of chemically induced developmental anomalies. Before me aningful data on the alteration of gene expression can be obtained, th e heterogeneity of normal expressions within a species must be evaluat ed. In situ hybridization is a powerful technique that can detect pert urbations in the spatial and/or temporal expression of genes. The hybr idization protocol described here utilizes P-33 end-labeled, single-st randed DNA oligomeric nucleotides. Some of the technical advantages to this approach are that (1) DNA probes are not sensitive to RNase, (2) the probes are based on published sequences and are chemically synthe sized, (3) probes are end-labeled utilizing a terminal transferase rea ction, producing probes of high specific activity and uniform length, (4) expression patterns of spliced genes can be easily evaluated by sy nthesizing probes specific to different areas of the gene relative to the splice site, (5) probes do not require the use of strong reducing agents, and (6) the technique can be used on tissue embedded in paraff in and avoids the use of frozen sections. The utility of this approach is demonstrated in this paper by the analysis of expression of Hoxa-7 during normal development of the CD-1 mouse embryo.