Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice.
Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.
Hagedorn Research Institute, Department of Developmental Biology, Niels Steensens Vej 6, DK-2820, Denmark.
Palle
P
Serup
Carsten
C
Gustavsen
Tino
T
Klein
Leah A
LA
Potter
Robert
R
Lin
Nandita
N
Mullapudi
Ewa
E
Wandzioch
Angela
A
Hines
Ashley
A
Davis
Christine
C
Bruun
Nina
N
Engberg
Dorthe R
DR
Petersen
Janny M L
JM
Peterslund
Raymond J
RJ
Macdonald
Anne
A
Grapin-Botton
Mark A
MA
Magnuson
Kenneth S
KS
Zaret
D028341:Activins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D005818:Genetic Engineering; D056426:Genetic Loci / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D022661:RNA, Untranslated; D051381:Rats; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000235:genetics
D019485:Bone Morphogenetic Proteins; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated; D051880:Receptors, Notch; C422405:activin A; D028341:Activins; D014212:Tretinoin