In vivo analysis of mRNA stability using the Tet-Off system in the chicken embryo.
The rate of mRNA degradation plays an important role in the control of gene expression. The mRNA stability is mainly dependent on cis-regulatory elements contained in the 3' or 5' untranslated region (UTR) of the mature mRNAs, and its regulation is an efficient way to adapt the level of a given transcript in the cell. Although this process has been well studied in cell culture, little is known about mRNA stability during embryonic development. Here, we describe an assay that combines the tetracyclin-dependent inducible system Tet-Off with in ovo electroporation to monitor mRNA stability in the chick neural tube. We show, by using the GFP intensity as an indirect reporter system, that the 3'UTR of Lunatic Fringe strongly destabilizes transcripts, while transcripts bearing the 3'UTR of Fgf8 are much more stable. This simple assay provides a powerful tool to study mRNA dynamics in vivo.
Stowers Institute for Medical Research, 1000E 50th, Kansas City, MO 64110, USA.
Valérie
V
Hilgers
Olivier
O
Pourquié
Julien
J
Dubrulle
D020413:3' Untranslated Regions; D000818:Animals; D030161:Avian Proteins; D018929:Cell Culture Techniques; D002642:Chick Embryo; D004318:Doxycycline / Q000494:pharmacology*; D018274:Electroporation; D047108:Embryonic Development; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D017930:Genes, Reporter; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D017403:In Situ Hybridization; D007700:Kinetics; D020871:RNA Stability / Q000187:drug effects*; D012333:RNA, Messenger / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D019076:Transgenes
D020413:3' Untranslated Regions; D030161:Avian Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors; D016695:Glycosyltransferases; C498394:LFNG protein, Gallus gallus; D004318:Doxycycline