title: Monitoring Long Interspersed Nuclear Element 1 Expression During Mouse Embryonic Stem Cell Differentiation.
abstract: Long Interspersed Elements-1 (LINE-1 or L1) are a class of transposable elements which account for almost 19 % of the mouse genome. This represents around 600,000 L1 fragments, among which it is estimated that 3000 intact copies still remain capable to retrotranspose and to generate deleterious mutation by insertion into genomic coding region. In differentiated cells, full length L1 are transcriptionally repressed by DNA methylation. However at the blastocyst stage, L1 elements are subject to a demethylation wave and able to be expressed and to be inserted into new genomic locations. Mouse Embryonic Stem Cells (mESCs) are pluripotent stem cells derived from the inner cell mass of blastocysts. Mouse ESCs can be maintained undifferentiated under controlled culture conditions or induced into the three primary germ layers, therefore they represent a suitable model to follow mechanisms involved in L1 repression during the process of differentiation of mESCs. This protocol presents how to maintain culture of undifferentiated mESCs, induce their differentiation, and monitor L1 expression at the transcriptional and translational levels. L1 transcriptional levels are assessed by real-time qRT-PCR performed on total RNA extracts using specific L1 primers and translation levels are measured by Western blot analysis of L1 protein ORF1 using a specific L1 antibody.
authors.affiliation: Department of Biology, Swiss Federal Institute of Technology, HPL G32.1, Otto-Stern-Weg 7, CH-8093, Zurich, Switzerland.
authors.forename: Maxime
authors.initials: M
authors.lastname: Bodak
authors.affiliation: Department of Biology, Swiss Federal Institute of Technology, HPL G32.1, Otto-Stern-Weg 7, CH-8093, Zurich, Switzerland. cciaudo@ethz.ch.
authors.forename: Constance
authors.initials: C
authors.lastname: Ciaudo
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D007150:Immunohistochemistry / Q000379:methods; D020084:Long Interspersed Nucleotide Elements; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
keywords: Embryonic body differentiation; LINE-1; Protein extraction and Western blot; RNA extraction and real-time qRT-PCR; mESCs