Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.
We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.
Semprini
Sabrina
S
University of Edinburgh, Queen's Medical Research Institute, Edinburgh EH16 4TJ, Scotland, UK.
Friedrichsen
Sonke
S
Harper
Claire V
CV
McNeilly
Judith R
JR
Adamson
Antony D
AD
Spiller
David G
DG
Kotelevtseva
Nina
N
Brooker
Gillian
G
Brownstein
David G
DG
McNeilly
Alan S
AS
White
Michael R H
MR
Davis
Julian R E
JR
Mullins
John J
JJ
D000818:Animals; D002460:Cell Line; D004967:Estrogens / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology; D008297:Male; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011916:Rats, Inbred F344; D055647:Rats, Transgenic
D004967:Estrogens; D008070:Lipopolysaccharides; D011388:Prolactin