Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Floxin, a resource for genetically engineering mouse ESCs.

ABSTRACT: We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.

SUBMITTER: Singla V

PROVIDER: S-EPMC2895430 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Floxin, a resource for genetically engineering mouse ESCs.

Singla Veena V Hunkapiller Julie J Santos Nicole N Seol Allen D AD Norman Andrew R AR Wakenight Paul P Skarnes William C WC Reiter Jeremy F JF

Nature methods 20091206 1

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs. ...[more]

PMID: 19966808

Similar Datasets

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

Project description:Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins.

| S-EPMC4491196 | biostudies-literature

Metastable pluripotent states in NOD-mouse-derived ESCs.

Project description:Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

| S-EPMC2714944 | biostudies-literature

Genetically Engineering a Susceptible Mouse Model for MERS-CoV-Induced Acute Respiratory Distress Syndrome.

Project description:Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330+/+ MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330+/+ mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330+/+ mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.

| S-EPMC7123801 | biostudies-literature

Engineering genetically encoded FRET sensors.

Project description:Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening.

| S-EPMC4168480 | biostudies-literature

A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.

Project description:The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

| S-EPMC4847940 | biostudies-literature

Depletion of nuclear LINE1 RNA in mouse ESCs and embryos.

Project description:LINE1 is the most active and abundant family of retrotransposons; it is implicated in a number of pathologies, as well as in early embryo development. We present a protocol to specifically knockdown LINE1 in mouse embryonic stem cells and embryos, including details for the nucleofection and zygote microinjection of LINE antisense oligos, followed by RNA FISH validation. This protocol can be used in development, as well as other cell types where LINE1 is believed to be expressed. For complete information on the use and execution of this protocol, please refer to Percharde et al. (2018).

| S-EPMC8361322 | biostudies-literature

Efficient generation of mouse ESCs-like pig induced pluripotent stem cells.

Project description: Not available

| S-EPMC3996154 | biostudies-literature

Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs.

Project description:In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

| S-EPMC9439721 | biostudies-literature

Mouse SCNT ESCs have lower somatic mutation load than syngeneic iPSCs.

Project description:Ectopic expression of reprogramming factors has been widely adopted to reprogram somatic nucleus into a pluripotent state (induced pluripotent stem cells [iPSCs]). However, genetic aberrations such as somatic gene mutation in the resulting iPSCs have raised concerns regarding their clinical utility. To test whether the increased somatic mutations are primarily the by-products of current reprogramming methods, we reprogrammed embryonic fibroblasts of inbred C57BL/6 mice into either iPSCs (8 lines, 4 previously published) or embryonic stem cells (ESCs) with somatic cell nuclear transfer (SCNT ESCs; 11 lines). Exome sequencing of these lines indicates a significantly lower mutation load in SCNT ESCs than iPSCs of syngeneic background. In addition, one SCNT-ESC line has no detectable exome mutation, and two pairs of SCNT-ESC lines only have shared preexisting mutations. In contrast, every iPSC line carries unique mutations. Our study highlights the need for improving reprogramming methods in more physiologically relevant conditions.

| S-EPMC3986627 | biostudies-literature

mRNA profiling of genetically matched ESCs and iPSCs

Project description:Induced pluripotent stem cells (iPSCs) can be generated by enforced expression of defined transcription factors in somatic cells. It remains controversial whether iPSCs are equivalent to blastocyst-derived embryonic stem cells (ESCs). Using genetically matched cells, we found that the overall mRNA expression patterns of these cell types are indistinguishable with the exception of a few transcripts encoded on chromosome 12qF1.

2010-04-28 | GSE20572 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data