A novel microarray strategy for detecting genes and pathways in microbes with unsequenced genomes

Expression profile analysis of genes provides valuable information concerning the genetic response of cells to stimuli. We describe an adaptation of this technology that can be used to probe for the expression of specific families of genes in microbial species. In our method a combination of sets of oligonucleotide probes representing fingerprint sequences specific to protein families is used to identify the presence and expression levels of family homologs in a microbial cell. We demonstrate computationally, using exemplars, that when the cDNA complement from an organism is sequentially screened against a set of specific motif oligonucleotides, statistically significant information can be obtained concerning the expression of the corresponding genes. This method can be used to identify specific genes and pathways simultaneously in several organisms of interest even in the absence of sequence information from the organisms.     

Suppression of gene expression by RNA interference in cultured plant cells

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.     

Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.     

A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.     

Murine Cell Glycolipids Customization by Modular Expression of Glycosyltransferases

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.     

Discriminative feature of cells characterizes cell populations of interest by a small subset of genes

Organisms are composed of various cell types with specific states. To obtain a comprehensive understanding of the functions of organs and tissues, cell types have been classified and defined by identifying specific marker genes. Statistical tests are critical for identifying marker genes, which often involve evaluating differences in the mean expression levels of genes. Differentially expressed gene (DEG)-based analysis has been the most frequently used method of this kind. However, in association with increases in sample size such as in single-cell analysis, DEG-based analysis has faced difficulties associated with the inflation of P-values. Here, we propose the concept of discriminative feature of cells (DFC), an alternative to using DEG-based approaches. We implemented DFC using logistic regression with an adaptive LASSO penalty to perform binary classification for discriminating a population of interest and variable selection to obtain a small subset of defining genes. We demonstrated that DFC prioritized gene pairs with non-independent expression using artificial data and that DFC enabled characterization of the muscle satellite/progenitor cell population. The results revealed that DFC well captured cell-type-specific markers, specific gene expression patterns, and subcategories of this cell population. DFC may complement DEG-based methods for interpreting large data sets. DEG-based analysis uses lists of genes with differences in expression between groups, while DFC, which can be termed a discriminative approach, has potential applications in the task of cell characterization. Upon recent advances in the high-throughput analysis of single cells, methods of cell characterization such as scRNA-seq can be effectively subjected to the discriminative methods.     

Gene delivery to adult neural stem cells

Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.     

Isolation of mRNA from specific tissues of Drosophila by mRNA tagging

To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Genetic modification of human embryonic stem cells for derivation of target cells

Directed differentiation of human embryonic stem cells (hESCs) may yield models to study organogenesis, produce cells and tissues for therapies, and identify clinically relevant compounds for disease treatment. Optimal conditions for specific differentiation of hESCs are still being determined. Incorporation of fluorescent reporter genes will enable high-throughput screening to identify fate-specifying molecules. Ectopic expression, or silencing, of key developmental genes can also direct differentiation toward specific lineages. Here, we briefly overview various genetic modifications used to generate useful hESC lines. We identify strengths and limitations to each method and propose the most suitable approaches for different applications.     

Determination of stromal signatures in breast carcinoma

Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.     

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.     

Stage-specific gene expression in erythroid progenitor cells (CFU-E)

In erythropoietic differentiation, mature red blood cells are generated from specific progenitor cells through the action of specific growth regulatory molecules. To know the mechanism of differentiation, it is important to examine the control of gene expression in these progenitor cells in combination with growth regulatory molecules. We have cloned two genes expressing at a maximal level in the CFU-E (colony forming unit-erythroid), one of the erythroid progenitor cells from novel murine erythroleukemia (MEL) cell line (TSA8) which can be induced to CFU-E in vitro. The expression of these genes is well correlated with the appearance of CFU-E during induction of TSA8 cells, and is higher in the CFU-E-cells enriched from mouse fetal livers than in the more differentiated erythroid cells. Combining these with our previous results, it is suggested that in the erythropoiesis the progenitor cells have distinct patterns of gene expression. This expression is replaced through each progenitor cell rather than by the continuous increase in the expression of a set of genes specific to the mature erythroid cell following the commitment process.