The oocyte is a key regulator of ovarian folliculogenesis and early embryonic development. However, the composition of the oocyte transcriptome and identities and functions of key oocyte-specific genes involved in the above processes are relatively unknown. Using a PCR-based cDNA amplification method (SMART technology), we constructed a bovine oocyte cDNA library. Analysis of 230 expressed sequence tags (ESTs) from this library identified 102 unique sequences. Although some correspond to housekeeping genes (e.g., ribosomal protein L15) and some represent genes previously known to be expressed in oocytes and other tissues, most encode for genes whose expression in mammalian oocytes has not been reported previously (e.g., cocaine- and amphetamine-regulated transcript) or genes of unknown function. Sixteen did not show significant sequence similarity to any entries in the GenBank database and were classified as novel. Using over 2,000 unsequenced, randomly selected cDNA clones from the library, we constructed an oocyte microarray and performed experiments to identify genes preferentially expressed in fetal ovary (an enriched source of oocytes) relative to somatic tissues. Eleven clones were identified by microarray analysis with consistently higher expression in fetal ovaries (collected from animals at days 210-260 of gestation) compared with spleen and liver. DNA sequence analysis of these clones revealed that two correspond to JY-1, a novel bovine oocyte-specific gene. The remaining nine clones represent five identified genes and one additional completely novel gene. Increased abundance of mRNA in fetal ovary for five of the six genes identified was confirmed by real-time PCR. Results demonstrate the potential utility of these unique resources for identification of oocyte-expressed genes potentially important for reproductive function.
Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study responses of thousands of genes to internal and external stimuli. In neurobiology, studies of the human brain have been expanding rapidly by use of functional genomics techniques. To enhance these studies and allow use of a porcine brain model, a normalized porcine brain cDNA library (PBL) has been generated and used as a base for EST discovery and microarray generation. In this report, we discuss initial sequence analysis of 965 clones from this resource. Our data revealed that library normalization successfully reduced the number of clones representing highly abundant cDNA species and overall clone redundancy. Cluster analysis revealed over 800 unique cDNA species representing a redundancy rate for the normalized library of 6.9% compared with 29.4% before normalization. Sequence information, BLAST results, and TIGR cluster matches for these ESTs are publicly available via a web-accessible database (http://nbfgc.msu.edu). A cDNA microarray was created using 877 unique porcine brain EST amplicons spotted in triplicate on glass slides. This microarray was assessed by performing a series of experiments designed to test hybridization efficiency and false-positive rate. Our results indicate that the PBL cDNA microarray is a robust tool for studies of brain gene expression using swine as a model system.
The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo aging for 4 h with or without added Dex. Additional neutrophils were cotreated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of National Bovine Functional Genomics Consortium (NBFGC) microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4-h (untreated) neutrophils. Subsequent two-step mixed-model analysis detected (P < or = 0.05) 1,109 differentially expressed genes, of which contrast analysis indicated those that were independently responsive to aging (1,064), Dex (502), RU486 + Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 did not cluster well based on known function, and 634 clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex-responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound-healing actions of glucocorticoids.
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