[show abstract][hide abstract] ABSTRACT: Glucocorticoid receptor (GR) is a steroid hormone receptor that has been shown to play important roles in diverse cellular and physiological processes. More and more evidence has revealed that the effects of glucocorticoids are mediated by the glucocorticoid receptor through genomic or nongenomic mechanisms. A growing number of glucocorticoid receptor splice variants have been identified in human tissues, but few are known in rat tissues. In this work, a novel rGR cDNA, called rGRbeta, was cloned from Sprague Dawlay (SD) rat liver. Sequence analysis revealed that the rGRbeta mRNA was 39 base pairs (bp) shorter than the rGR mRNA reported earlier. The deleted segment is located in exon 1 and encodes 13 repeated glutamine residues. Both the rGR and rGRbeta mRNAs were quantitated by Northern blot hybridization using non-homologous glucocorticoid cDNA probes. Results showed that the rGR and rGRbeta mRNAs were most abundant in the lung, the least abundant in the heart, and there were more rGR and rGRbeta mRNAs in the kidney than in the liver. The identification of rGRbeta may contribute to the understanding of the genomic or nongenomic effects of glucocorticoids.
[show abstract][hide abstract] ABSTRACT: Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 05/2006; 23(2):396-9.
[show abstract][hide abstract] ABSTRACT: The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.
Molecular Reproduction and Development 03/2002; 61(2):164-72. · 2.81 Impact Factor