Dataset Information

Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.

ABSTRACT: DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38 degrees C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 microg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.

SUBMITTER: Giraldo AM

PROVIDER: S-EPMC6042044 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.

Giraldo Angelica M AM Vaught Todd D TD Fu Limin L Duncan Alison J AJ Vance Amy M AM Mendicino Michael M Ayares David L DL

Gene expression 20090101 5

DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA an ...[more]

PMID: 19630269

Similar Datasets

Project description:OBJECTIVE:Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. METHODS:We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Ppar?2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. RESULTS:CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Ppar?2, was successfully achieved using selected sgRNAs targeting the Ppar?2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Ppar?2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. CONCLUSIONS:We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.

Project description:siRNA treatment has great promise to specifically control gene expression and select cell behaviors but has delivery challenges limiting its use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effectively deliver siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA. This is vastly superior to a commercially available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), was cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT/siRNA treatment resulted in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There was no statistically significant differences on cell surface markers of diblock copolymer/siRNA or DharmaFECT/siRNA-treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there was no discernible differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitated uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC survival or differentiation capacity and therefore show great promise for use within regenerative medicine applications.

Dataset Information

Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.

Publications

Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets