An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles

Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (τ), quantitative trait locus (QTL) analysis was performed using F2 mice obtained from an F1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For τ, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes

Delay in feedback repression by Cryptochrome 1 is required for circadian clock function

Direct evidence for the requirement of delay in feedback repression in the mammalian circadian clock has been elusive. Cryptochrome 1 (Cry1), an essential clock component, displays evening-time expression and serves as a strong repressor at morning-time elements (E box/E′ box). In this study, we reveal that a combination of day-time elements (D box) within the Cry1-proximal promoter and night-time elements (RREs) within its intronic enhancer gives rise to evening-time expression. A synthetic composite promoter produced evening-time expression, which was further recapitulated by a simple phase-vector model. Of note, coordination of day-time with night-time elements can modulate the extent of phase delay. A genetic complementation assay in Cry1−/−:Cry2−/− cells revealed that substantial delay of Cry1 expression is required to restore circadian rhythmicity, and its prolonged delay slows circadian oscillation. Taken together, our data suggest that phase delay in Cry1 transcription is required for mammalian clock function

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.</p

Feedback repression is required for mammalian circadian clock function

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function. Circadian clocks have been proposed to consist of autoregulatory loops that use transcriptional feedback and regulated protein turnover to maintain 24-h periodicity 1-3 . However, the universal requirement for transcriptional feedback repression has been questioned recently by two studies showing that it is not required for circadian rhythms in cyanobacteria 4,5 . Circadian feedback repression in mammals is believed to be mediated by the CRY1 and CRY2 and PERIOD (PER1 and PER2) proteins. CRY and PER are hypothesized to autoregulate their own expression by repressing the heterodimeric complex of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) domain transcriptional activators CLOCK and BMAL1, which bind to E-box elements in the CRY 6 and PER 7,8 promoters. Indirect support for this feedback mechanism comes from biochemical, molecular and genetic evidence: (i) CRY and PER physically interact with CLOCK/BMAL1, (ii) CRY and PER repress CLOCK/BMAL1 activity in steady-state transcriptional assays 9-12 and (iii) CRY and PER genes are required for maintenance of circadian rhythms in mice RESULTS Mutagenesis and functional screening of CLOCK In order to determine the requirement of feedback repression in circadian clock function, we sought to identify CLOCK alleles that were insensitive to CRY1 repression but that maintained normal transcriptional activity. We generated a random library of B6,000 mutant human CLOCK alleles. We screened clones individually in cell-based reporter assays with wild-type BMAL1 cDNA and a PER1 promoterluciferase construct 7 in the presence of cotransfected CRY1. Of the approximately 6,000 CLOCK clones screened, three clones (Clock-1, Clock-2 and Clock-3) reproducibly maintained threefold or greater reporter activity in the presence or absence of CRY1 compared with wild-type CLOCK. We verified the reduced CRY1 sensitivity phenotype by 96-well assays of activity of CLOCK mutants with increasing amounts of CRY1 plasmid. When cotransfected with wild-type BMAL1 and the PER1 reporter, the Clock-1, Clock-2 and Clock-3 clones maintained noticeably greater activity than wild-type CLOCK in the presence of 1 or 2.5 ng of CRY1 plasmi

Direct application of the newly developed method to single ICM cells from mouse E3

<p><b>Copyright information:</b></p><p>Taken from "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis"</p><p>Nucleic Acids Research 2006;34(5):e42-e42.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409679.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p>5 blastocyst reveals the presence of two distinct cell populations. () Hierarchical clustering of single ICM cells. () Heat map representation of differentially expressed genes (top 100). The expression levels are color-coded from red (high) to blue (low). The expression levels are normalized in the lows. () The correlation of gene expression is preserved between E3.5 and E4.5. The copy numbers of expressed genes were estimated with Q-PCR. Orange, pink and green bars represent high, middle and low/non-detectable expression of , respectively. -values of the Chi-square test for independence from expression are indicated. (D and F) Blastocysts at E3.5 () and E4.5 (). The typical embryos used for single-cell experiments are shown. (E and G) Expression levels of key genes related to PE and epiblast at E3.5 () and E4.5 (). All of the single-cell samples of ICMs are shown. The representation code is the same as in (C)