Single-cell Gene Research Articles

A hierarchical model of transcriptional dynamics allows robust estimation of transcription rates in populations of single cells with variable gene copy number

Motivation: cis-regulatory DNA sequence elements, such as enhancers and silencers, function to control the spatial and temporal expression of their target genes. Although the overall levels of gene expression in large cell populations seem to be precisely controlled, transcription of individual genes in single cells is extremely variable in real time. It is, therefore, important to understand how these cis-regulatory elements function to dynamically control transcription at single-cell resolution. Recently, statistical methods have been proposed to back calculate the rates involved in mRNA transcription using parameter estimation of a mathematical model of transcription and translation. However, a major complication in these approaches is that some of the parameters, particularly those corresponding to the gene copy number and transcription rate, cannot be distinguished; therefore, these methods cannot be used when the copy number is unknown.Results: Here, we develop a hierarchical Bayesian model to estimate biokinetic parameters from live cell enhancer–promoter reporter measurements performed on a population of single cells. This allows us to investigate transcriptional dynamics when the copy number is variable across the population. We validate our method using synthetic data and then apply it to quantify the function of two known developmental enhancers in real time and in single cells.Availability: Supporting information is submitted with the article.Contact: d.j.woodcock@warwick.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Stem cells, pluripotency and nuclear reprogramming

The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. However, major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk.During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells and impeded identification of events at the single‐cell level. We utilized two different gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. We show that Esrrb, Utf1, Lin28, and Dppa2 have a better potential to predict cells that will become iPSCs, compared to Fbxo15, Fgf4, and Oct4, which were previously suggested to be markers of reprogramming. Our data suggest that stochastic gene expression early in reprogramming is followed by a late sequential phase with Sox2 activation upstream in a gene expression hierarchy. Finally, we demonstrate that subsets of downstream factors derived from the sequential phase can activate the pluripotency circuitry.A major impediment in realizing the potential of ES and iPS cells to study human diseases is the inefficiency of gene targeting. Using Zn finger or TALEN mediated genome editing we have established efficient protocols to target expressed and silent genes in human ES and iPS cells. Finally, our progress in using iPS cells for therapy and for the study of complex human diseases will be summarized.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50 pg total RNA, or 0.01-2.5 pg mRNA, per cell) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: greater numbers of genes to be analyzed per cell; more quantitative analysis of gene expression; and better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

Analysis of Affinity Maps of Membrane Proteins on Individual Human Embryonic Stem Cells

The heterogeneity found in many cell types has greatly inspired research in single-cell gene and protein profiling for discovering the origin of heterogeneity and its role in cell fate decisions. Among the existing techniques to probe heterogeneity, atomic force microscopy (AFM) utilizes an antibody/ligand-modified tip to explore the distribution of a target membrane protein on individual cells in their native environment. In this paper, we establish a practical model to analyze the data systematically, and attempt the quantification of membrane protein abundance on single cells by taking account issues, such as the level of nonspecific interaction, the probe resolution, and the reproducibility of detecting protein distribution. We demonstrated the application in examining the heterogeneous distribution and the local protein abundance of TRA-1-81 antigen on human embryonic stem (hES) cells at the subcellular level. Heterogeneity in TRA-1-81 expression was also detected at the single cell level, suggesting the presence of subpopulation cells within an undifferentiated hES cell colony. The method provides a platform to unveiling the correlation between heterogeneity of membrane proteins and cell development in a complex cell community.

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Somatic cell mutations in cerebral tissue of cattle affected by bovine spongiform encephalopathy

In animals the prion disease includes sheep and goat scrapie and the bovine spongiform encephalopathy (BSE). While several polymorphisms of the prion (PRNP) gene have been identified in sheep and some of them have been associated with susceptibility to scrapie, few mutations are reported in cattle and no correlation with BSE have been demonstrated. Genetic screening for mutants in the PRNP gene of 21 BSE positive animals by direct sequencing of the amplified gene, using DNA extracted from brain as template, confirmed that only few polymorphisms are present. However DNA molecules cloned and sequenced from the population of fragments considering a total of 90 clones from 9 BSE positive and 70 clones from 7 BSE negative animals, gave a highly significant differences in the frequency of mutations (p = 0.01). The high frequency and type of variants found cannot be explained only with misincorporation error of the Taq polymerase. Interestingly one of the mutations found in the BSE positive animals (F209S) corresponds to a mutant that causes a familiar form of prion disease in humans (F198S). These data can be explained with the presence of somatic mutations modifying the PRNP gene in single brain cells.

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells

Using sequential RNA–DNA fluorescence in situ hybridization, the nuclear arrangement of both the active and inactive c-myc gene as well as its transcription was investigated in colon cancer HT-29 cells induced to differentiate into enterocytes. Cytogenetic studies revealed the presence of two chromosomes 8 in HT-29 cells, of which the one containing c-myc gene amplicons was substantially larger and easily distinguished from the normal chromosome. This observation enabled detection of both activity and nuclear localization of c-myc genes in single cells and in individual chromosome territories. Similar transcriptional activity of the c-myc gene was observed in both the normal and derivative chromosome 8 territories showing no influence of the amplification on the c-myc gene expression. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography.

High-Resolution Imaging of Bone Precursor Cells Within the Intact Bone Marrow Cavity of Living Mice

Bone precursor cells (BPCs) play a critical role in bone maintenance and regeneration. Currently, no tool exists to study BPCs or other bone marrow cell types directly within their complex microenvironment. Here, we describe in vivo magnetic resonance imaging (MRI) of anatomical structures inside the medullary cavity of the mouse femur. We demonstrate that BPCs passively labeled with iron oxide-containing particles can be monitored by MRI within the intact bone marrow at an in-plane resolution of 43x25 microm. Anatomical detail provided by MRI is complemented by functional optical imaging of reporter gene expression. Single-cell dual iron oxide-reporter gene labeling has potential for combined cell tracking and cell biology studies. In summary, we describe a versatile platform suitable for studying the biology of many bone marrow cell types in living bone.

Multiple mutation analysis of the cystic fibrosis gene in single cells

PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.

GnRH and gpcr: laser-captured single cell gene profiling

We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.

Noise Propagation in Gene Networks

Accurately predicting noise propagation in gene networks is crucial for understanding signal fidelity in natural networks and designing noise-tolerant gene circuits. To quantify how noise propagates through gene networks, we measured expression correlations between genes in single cells. We found that noise in a gene was determined by its intrinsic fluctuations, transmitted noise from upstream genes, and global noise affecting all genes. A model was developed that explains the complex behavior exhibited by the correlations and reveals the dominant noise sources. The model successfully predicts the correlations as the network is systematically perturbed. This approach provides a step toward understanding and manipulating noise propagation in more complex gene networks.

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics