A strategy for the analysis of gene expression during neural development.
The delineation of cis-acting elements regulating stage-specific gene expression has played a critical role in the definition of the transcriptional circuitry regulating differentiation. In complex tissues such as the central nervous system, differentiation often requires temporally and spatially dynamic epigenetic cues that cannot be reproduced in cell culture. In these cases, identification of critical regulatory sequences and subsequent characterization of cognate transcription factors have been limited by lack of a rapid and efficient assay system for gene expression. We report a methodology that combines particle-bombardment transfection procedures and organotypic slice culture techniques to provide an acute assay system for transcriptional control in the developing central nervous system. Using this system, we demonstrate that cell-specific regulation of the brain lipid-binding protein (BLBP) gene in Bergmann glia, astrocytes, and migrating granule cells is conferred by 1.7 kb of 5' flanking sequences and that Purkinje cell-specific expression of the calbindin D28k gene in cerebellar cortex can be achieved with 1.1 kb of flanking DNA. Nearly 100% cotransfection of multiple DNAs can be achieved, allowing the design of precisely internally controlled experiments and providing the potential for rapid and efficient genetic analysis of gene function in single cells in a wild-type environment.
Arnold
D
D
Laboratory of Molecular Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Feng
L
L
Kim
J
J
Heintz
N
N
D000375:Aging; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000502:physiology; D015415:Biomarkers / Q000032:analysis; D064092:Calbindin 1; D064026:Calbindins; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015870:Gene Expression; D005786:Gene Expression Regulation; D007395:Interneurons / Q000166:cytology / Q000502:physiology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic; D011689:Purkinje Cells / Q000166:cytology / Q000502:physiology; D064030:S100 Calcium Binding Protein G / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
D015415:Biomarkers; C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D002352:Carrier Proteins; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D001616:beta-Galactosidase