Use of a reverse transcriptase-polymerase chain reaction assay to analyze allele-specific expression in individual hippocampal neurons.
We report here a single-cell RT-PCR assay for allele-specific gene expression that can be used to probe for somatic variability within the CNS. Such variability, arising from epigenetic (nonmutational) events or somatic mutation early in development, may give clues as to clonal origin and may also affect the inheritance pattern of some CNS disorders. As a model system, we used reciprocal F1 hybrids of the cross Mus musculus C57BL/6J x Mus musculus castaneus. RNA was isolated from individual dissociated pyramidal neurons from hippocampi of F1 pups. For each gene of interest, single base polymorphisms were identified between the two parental strains by automated sequencing of RT-PCR products. Allele-specific expression was then analyzed by means of the previously described quantitative RT-PCR single nucleotide primer extension (SNuPE) assay (Singer-Sam et al., PCR Methods Appl. 1:160-163, 1992). Individual neurons showed monoallelic expression of the two control genes, X-linked Rps4, and the imprinted gene Snrpn; in contrast expression of Ncam and F3cam, coding for neural cell adhesion molecules, was found to be biallelic.
Shen
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Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Gao
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Singer-Sam
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D000483:Alleles; D000818:Animals; D001324:Autoantigens / Q000235:genetics; D002678:Chimera; D003433:Crosses, Genetic; D005260:Female; D015870:Gene Expression; D006651:Histocytochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics*; D019006:Neural Cell Adhesion Molecules / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017966:Pyramidal Cells / Q000166:cytology*; D017411:Ribonucleoproteins, Small Nuclear; D012269:Ribosomal Proteins / Q000235:genetics; D055517:snRNP Core Proteins
D001324:Autoantigens; D009419:Nerve Tissue Proteins; D019006:Neural Cell Adhesion Molecules; D017411:Ribonucleoproteins, Small Nuclear; D012269:Ribosomal Proteins; C031798:ribosomal protein S4; D055517:snRNP Core Proteins