RT-PCR-based method to localize the spatial expression of genes in the mouse blastocyst.
We report an RT-PCR-based assay to analyze the spatial pattern of expression of genes in mouse blastocysts. The assay is based on comparing the amount of an amplicon for a specific gene in isolated inner cell mass cells to that in intact blastocysts and using this ratio to calculate the relative amount of transcript per inner cell mass cell or trophectoderm cell. Validation of the assay is demonstrated by documenting that the level of fgf-4 transcripts in inner cell mass cells is approximately 13 times greater than that in trophectoderm cells, and that the level of endo A transcripts in trophectoderm cells is approximately 18 times greater than that in inner cell mass cells; results of others have shown that fgf-4 and endo A expression are enhanced in the inner cell mass cells and trophectoderm cells, respectively. Using this assay, we find that transcripts for both the EGF receptor and TGF-alpha are present in both the inner cell mass and trophectoderm cells and that on a per cell basis, essentially equal amounts of each transcript are present in these two cell types. We also demonstrate by laser-scanning confocal microscopy that TGF-alpha protein is uniformly present in all cells of the blastocyst.
Brison
D R
DR
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA.
Schultz
R M
RM
D000818:Animals; D001757:Blastomeres / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000235:genetics*; D015870:Gene Expression; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D016211:Transforming Growth Factor alpha / Q000235:genetics*
C496446:Fgf4 protein, mouse; D051519:Fibroblast Growth Factor 4; D011518:Proto-Oncogene Proteins; D016211:Transforming Growth Factor alpha; D005346:Fibroblast Growth Factors; D066246:ErbB Receptors; D012194:RNA-Directed DNA Polymerase