Assessment and application of laser microdissection for analysis of gene expression in the rhesus monkey endometrium.
We investigated the use of laser capture microdissection (LCM) to identify differences in gene expression between cell types or regions within the rhesus monkey endometrium. Different cell types were harvested from the two major regions of the endometrium during midsecretory phases (Days 21-23) of adequate artificial menstrual cycles: glandular epithelia (G) or stroma (S) from the functionalis (F) or the basalis (B). Amplification of the cDNA populations (primer-specific adaptors) was used to increase the amount of nucleic acid for further analysis. This single amplification step allowed us to detect the housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA) and the cDNA smears in the samples. Using differential display reverse transcription polymerase chain reaction (DDRT-PCR), six fragments were selected, cloned, and sequenced based on their regional and cell type localization. Primer-specific PCR analysis subsequently confirmed the localization of three fragments: F1, highly expressed in the functionalis but not the basalis, was homologous (93% identical) to the human leukotriene B4 receptor BLT2; FS-1, highly expressed only in the stroma of the functionalis, had a 94% homology with an as yet uncharacterized gene (FLJ124360); and BG-1, primarily expressed only in the glandular epithelia of the basalis, showed a 98% homology with an uncharacterized bacterial artificial chromosome clone sequence. These LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell type- or region-specific gene expression in the rhesus monkey endometrium.
Torres
Mira S T
MS
Division of Endocrinology, Department of Obstetrics and Gynecology, University of Massachusetts Medical School, Worcester 01655, USA.
Ace
Christopher I
CI
Okulicz
William C
WC
D000818:Animals; D019703:Calcineurin / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D004343:Drug Implants; D004717:Endometrium / Q000378:metabolism* / Q000648:ultrastructure; D004967:Estrogens / Q000008:administration & dosage; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006637:Histamine N-Methyltransferase / Q000235:genetics; D016591:Histocytological Preparation Techniques; D006801:Humans; D007834:Lasers; D007975:Leukotriene B4 / Q000235:genetics; D008253:Macaca mulatta; D008597:Menstrual Cycle; D011374:Progesterone / Q000008:administration & dosage; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013048:Specimen Handling / Q000379:methods; D014018:Tissue Distribution
D018076:DNA, Complementary; D004343:Drug Implants; D004967:Estrogens; D007975:Leukotriene B4; D011374:Progesterone; D006637:Histamine N-Methyltransferase; D019703:Calcineurin