Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

Azithromycin Treatment Alters Gene Expression in Inflammatory,Lipid Metabolism,and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides.     

Androstenediol analogs as ER-beta-selective SERMs

A series of 19-substituted androstenediol derivatives was prepared. Some of the novel analogs were surprisingly potent and selective ligands for ER-beta.     

Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection

Background

The CombiMatrix ElectraSense® microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs.

Methodology/Principal Findings

An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense® microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.

Conclusions/Significance

Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.     

Repairing breaks in the plant genome: the importance of keeping it together

DNA damage threatens the integrity of the genome and has potentially lethal consequences for the organism. Plant DNA is under continuous assault from endogenous and environmental factors and effective detection and repair of DNA damage are essential to ensure the stability of the genome. One of the most cytotoxic forms of DNA damage are DNA double-strand breaks (DSBs) which fragment chromosomes. Failure to repair DSBs results in loss of large amounts of genetic information which, following cell division, severely compromises daughter cells that receive fragmented chromosomes. This review will survey recent advances in our understanding of plant responses to chromosomal breaks, including the sources of DNA damage, the detection and signalling of DSBs, mechanisms of DSB repair, the role of chromatin structure in repair, DNA damage signalling and the link between plant recombination pathways and transgene integration. These mechanisms are of critical importance for maintenance of plant genome stability and integrity under stress conditions and provide potential targets for the improvement of crop plants both for stress resistance and for increased precision in the generation of genetically improved varieties.     

The role of hormonal therapy in osteoporosis

In developed societies, the post-menopausal period covers approximately one third of a woman's life. The deficit of oestrogens observed during the post-menopausal period significantly affects the course of many metabolic processes, causing a number of diseases and in consequence diminishing quality of life. Among others, bones belong to oestrogen-dependent tissues. The deficit of the protective influence of oestrogens compromises the dynamic balance of the bone transformation process towards resorption, thus reducing bone mass and quality, while increasing the risk of low-energy fractures. In recent years, differing views on the application of oestrogen/gestagen therapy have reached the level of controversy. The results of numerous clinical studies are far from unequivocal, with the whole subject one of heated debate. It has been confirmed that hormonal therapy prevents bone quality deterioration, while opening a protective umbrella around the bone, reducing the risk of osteoporotic fractures. A rational approach to weighing possible advantages against possible risks and a thorough evaluation of a patient's health condition allows for optimal therapy selection.     

The role of anorexia nervosa in secondary osteoporosis development with the risk for low energy fractures

Anorexia nervosa (AN) has in recent years become considerably more common. The disease primarily affects girls and young women, and also boys and young men. AN is a risk factor for secondary osteoporosis. AN-related metabolic disturbances lead to diminished bone quality and increased risk of fractures. The consequences of low energy fractures are the main causes of death in women with AN. Hormonal disturbances (e.g. hypoestrogenism, increased levels of ghrelin and Y peptide, changes in leptin and endocannabinoid levels), as well as the mechanisms involved in bone resorption (RANK/RANKL/OPG), are considered to be of great importance for anorectic bone quality. The risk of osteoporotic, non-vertebral fractures in AN patients is significantly higher than in healthy women. An improvement of bone mineral density is possible after substantial body mass increase. Weight loss, in conjunction with a well-balanced, controlled diet, is the key to correct peak bone mass levels, and diminishes the risk of osteoporosis with its consequence of low energy bone fractures. (Pol J Endocrinol 2011; 62 (1): 45-47).