Complementary DNA synthesis in situ: methods and applications.
In situ transcription is the synthesis of cDNA within cells. This chapter has illustrated some of the application of IST to the study of gene expression in complex cell environments. While the importance of transcription in modulating cellular activity has been long appreciated, the role of translational control mechanisms in regulating central nervous system functioning is just beginning to be recognized. Previous limitations in the availability of tissue have made it difficult to construct cDNA libraries from defined cell populations, to examine translational control, and to quantitate differences in the amount of mRNA for many distinct mRNAs in the same sample. In situ transcription facilitates all of these procedures, making it possible to characterize aspects of gene regulation that were previously difficult. Indeed, taken to its furthest extreme it is now possible to characterize gene expression in single live cells. This level of analysis allows basic questions, such as How different morphologically identical cells are at the level of gene expression, and How synaptic connectivity and glial interactions influence gene expression in single cells, to be experimentally approached. The ability to characterize gene expression in small amounts of tissue and single cells is critical to gaining an understanding of the contribution of specific cell types to the physiology of the central nervous system.
Eberwine
J
J
Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia 19104.
Spencer
C
C
Miyashiro
K
K
Mackler
S
S
Finnell
R
R
D000818:Animals; D001345:Autoradiography / Q000379:methods; D015139:Blotting, Southern / Q000379:methods; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D004622:Embryo, Mammalian; D017403:In Situ Hybridization / Q000379:methods; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010902:Pituitary Gland / Q000378:metabolism; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic
D003845:Deoxycytosine Nucleotides; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D016372:RNA, Antisense; D012333:RNA, Messenger; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA