220-plex microRNA expression profile of a single cell

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.     

Quantitative high-resolution genomic analysis of single cancer cells

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.     

Molecular characterization,expression pattern,and functional analysis of the <Emphasis Type="Italic">OsIRL</Emphasis> gene family encoding intracellular Ras-group-related LRR proteins in rice

Leucine-rich repeat proteins constitute a large gene family and play important roles in plant growth and development. Among them, Arabidopsis PIRL is a plant-specific class of intracellular Ras-group-related leucine-rich repeat proteins. In this study, we identified eight homologues of PIRLs in rice and designated them as OsIRL proteins. We described the gene structures, chromosome localizations, protein motifs, and phylogenetic relationships of the OsIRL gene family. The expression profiles of OsIRL genes were analyzed throughout the entire rice life cycle, along with light and three hormone stress conditions, using quantitative RT-PCR and microarray data. All OsIRL genes were expressed in at least one experimental stage and exhibited divergent expression patterns, with several genes showing preferential expression at specific stages. OsIRL4 and OsIRL5 showed higher expression levels under light compared to dark. OsIRL4 and OsIRL7 exhibited significant differential expression in response to hormone treatments. Six T-DNA or Tos17 insertion lines for five individual OsIRL genes were identified and examined morphologically. The comprehensive expression profile elucidated in this investigation together with the characterized insertion lines will provide a solid foundation for in-depth dissection of OsIRL functions.     

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.     

棉花纤维发育相关基因GhPRP10的克隆及其功能分析

利用电子序列拼接结合RT-PCR技术,从12DPA(开花后天数)棉纤维中克隆到1个编码富含脯氨酸蛋白(PRPs)基因,命名为GhPRP10(登录号KP036633)。GhPRP10基因开放阅读框为684bp,编码228个氨基酸,其中脯氨酸(Pro)含量为34.6%。序列分析发现GhPRP10蛋白具有N端信号肽和富含脯氨酸区域,属于第一类PRPs。实时荧光定量PCR(RT-PCR)结果显示,GhPRP10在棉纤维组织中优势表达,在纤维发育过程中的表达量呈现先升高后降低的趋势,在18DPA纤维中表达量最高。利用Gateway技术构建植物过量表达载体,转入烟草BY-2悬浮细胞,表型观察和细胞长度测量结果显示,转GhPRP10基因细胞比野生型细胞显著增长。根据该基因的组织表达特征和转基因细胞表型分析,推测GhPRP10基因在纤维伸长和次生壁合成过程中发挥作用。     

Gene fishing: the use of a simple protocol to isolate multiple homeodomain classes from diverse invertebrate taxa

Abstract Comparison of relevant gene sequence and functional data is central to understanding the evolution of metazoan development. The conservation of portions of regulatory genes, such as homeoboxes, allows for the design of PCR-based sequence isolation and amplification strategies. Here we describe a simple protocol that uses a degenerate primer pair to isolate a variety of homeobox-containing genes from diverse metazoan taxa. In a nonexhaustive survey, we have isolated 28 gene sequence fragments from 15 taxa, representing eight invertebrate phyla (Mollusca, Echiura, Annelida, Platyhelminth, Acoela, Ctenophora, Cnidaria, and Porifera). Based on BLAST and parsimony analyses, these gene fragments affiliate with several gene groups (PAIRED-like, HOX, and ParaHOX) and several single genes, including pancreas/duodenum homeoboxes (Pdx), empty spiracles (ems/Emx), gastrulation brain homeoboxes (Gbx), hematopoietically expressed homeoboxes (HEX), brain specific homeobox (bsh/BarH1/BarH2), NK-1 (NK-1/s59/slouch), and ladybird (Lbl/Lbe/Lbx). In several cases, these fragments represent the first reported orthologue for the phylum or superphyletic group (i.e., Lophotrochozoa).     

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications¹. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context². In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences³. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology—provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications—for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.     

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Specific proteomic adaptation to distinct environments in Vibrio parahaemolyticus includes significant fluctuations in expression of essential proteins

Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analysing the proteome of our model organism Vibrio parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony, there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programmes that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life-style, environment and differentiation state of the bacterium.     

Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures.

A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells; a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of fluorescently labelled oligonucleotide probes to rRNA. The method allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells.     

Transfection,Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.     

Screening for candidate genes involved in tolerance to organic solvents in yeast

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents.