This Title All WIREs
How to cite this WIREs title:
WIREs Dev Biol
Impact Factor: 5.814

Freedom of expression: cell‐type‐specific gene profiling

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single‐cell expression profiling. WIREs Dev Biol 2014, 3:429–443. doi: 10.1002/wdev.149 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Technologies > Analysis of the Transcriptome
Cell/nucleus isolation and profiling without cell isolation: two contrasting approaches. (a) Cell/nucleus isolation. Specific cells or nuclei of interest are identified and isolated from the host tissue. Total DNA/RNA/protein is extracted. Immuno‐ or genetic labeling may be required for identification. (b) Profiling without cell isolation. A labeling construct is expressed in a cell‐type‐specific manner in vivo. Material is labeled only in these cells. The whole tissue is homogenized and labeled molecules purified for identification.
[ Normal View | Magnified View ]
Targeted DamID (TaDa). TaDa identifies actively transcribed genes throughout the genome. A translational fusion between Dam methyltransferase and RNA polymerase II subunit (Dam–Pol II) is expressed in target cells. Pol II allows recruitment to active loci, and Dam specifically methylates adenine residues in local GATC sequences. At the desired stage, the whole tissue is homogenized and methylated genomic DNA is digested using DpnI restriction endonuclease. Selective amplification through the polymerase chain reaction (PCR) allows sequencing of these fragments.
[ Normal View | Magnified View ]
TU tagging. TU tagging labels nascent RNAs. Uracil phosphoribosyltransferase (UPRT) enzyme is expressed specifically in the cell type of interest. 4‐Thiouracil (4‐TU) is administered to the organism at the time point of interest through injection, bath incubation or feeding. UPRT incorporates 4‐TU into newly synthesized transcripts. The tissue is homogenized and thio‐RNAs are extracted, conjugated with biotin, and selectively purified using streptavidin‐coated beads.
[ Normal View | Magnified View ]
Cell‐type‐specific RNA co‐immunoprecipitation approaches. All approaches involve cell‐type‐specific expression of an epitope‐tagged RNA‐binding protein. At the desired time, the tissue is disrupted and protein–RNA complexes selectively isolated from the total lysate using antibodies directed against the tag. Protease treatment removes proteins, leaving purified RNA for amplification and/or sequencing. (a) mRNA tagging employs tagged PABP to isolate poly(A) RNAs. (b) TRAP/RiboTag utilizes a tagged ribosome subunit to precipitate actively translated mRNAs in ribosome/polysome complexes. (c) miRAP makes use of a tagged Argonaute family protein to co‐purify associated miRNAs.
[ Normal View | Magnified View ]
Nucleus sorting using Isolation of nuclei tagged in specific cell types (INTACT) and Batch isolate tissue‐specific chromatin for immunoprecipitation (BiTS‐ChIP). (a) The INTACT system. A nuclear targeting fusion (NTF) protein and a biotin ligase, BirA, are co‐expressed in cells of interest. The NTF protein is targeted to the nuclear membrane, where it acts as a substrate for BirA. After tissue homogenization, biotinylated nuclei are recovered using streptavidin‐coated magnetic beads in a magnet‐activated cell sorting (MACS)‐like protocol. (b) The BiTS‐ChIP system. Nuclei of interest are labeled fluorescently through cell‐type‐specific expression of fluorescent histone proteins. Alternatively, epitope‐tagged histones can be expressed and fluorescent antibodies are used against the tag (as shown). Following tissue dissociation, fluorescent nuclei are sorted using fluorescence‐activated cell sorting (FACS). MS, mass spectrometry.
[ Normal View | Magnified View ]
Cell sorting using fluorescence‐activated cell sorting (FACS) and magnet‐activated cell sorting (MACS). (a) FACS sorts cells on the basis of fluorescence. Examples of labeling methods include genetic expression of a fluorescent protein or, following dissociation, incubation with a fluorophore‐conjugated antibody to an endogenous antigen. Single cells are sorted sequentially based on user‐defined parameters including fluorescence intensity, size, and shape. The purified cells can be used for DNA, RNA, or protein studies. (b) MACS is an affinity purification approach to cell sorting. A cell‐type‐specific surface epitope must be present, or an exogenous antigen provided transgenically. Antibody‐coated magnetic beads capture target cells and can themselves be isolated with a magnetic rack. Trapped cells are released and may be used for DNA, RNA, or protein studies. MS, mass spectrometry.
[ Normal View | Magnified View ]

Browse by Topic

Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics
Technologies > Analysis of the Transcriptome