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.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

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.     

Antepartum glucose tolerance test results as predictors of type 2 diabetes mellitus in women with a history of gestational diabetes mellitus: A systematic review

Background: Women with a history of gestational diabetes mellitus (GDM) are at high risk for type 2 diabetes mellitus (T2DM).Objective: We reviewed prospective studies of antepartum glucose tolerance test results as risk factors for development of T2DM among women with a history of GDM.Methods: We searched 4 electronic databases and hand-searched 13 journals for literature published through January 2007. The search strategy consisted of medical subject headings and text words for GDM, T2DM, and other relevant terms. Articles were excluded for the following reasons: (1) not written in English; (2) no human data; (3) no original data; (4) <90% of sample was diagnosed with GDM without a separate analysis for women with GDM; (5) case report or series; (6) diagnosis of GDM not based on 3-hour 100-g oral glucose tolerance test (OGTT) or 2-hour 75-g OGTT; (7) T2DM not evaluated as outcome; (8) no relative measure of association or incidence reported; or (9) design did not address antepartum OGTT as a predictor of T2DM. Two investigators independently reviewed citations, performed serial data abstraction on full articles, and assessed the quality of each article. Data were abstracted for study participants and characteristics, T2DM diagnosis, length of follow-up, regression model covariates, and measures of association and variability.Results: Of 11,400 unique citations, we identified 11 articles that evaluated antepartum glucose testing and risk of T2DM in women with a history of GDM. Five studies found that the fasting blood glucose (FBG) on the antepartum diagnostic OGTT was a significant predictor of T2DM (odds ratio [OR] range: 11.1–21.0; relative risk [RR] range: 1.37–1.5; relative hazard [RH] = 2.47). Risk of incident T2DM was predicted by the antepartum 2-hour OGTT plasma glucose in 3 studies (OR range: 1.02–1.03; RR = 1.3) and by the antepartum OGTT glucose AUC in 3 other studies (OR range: 3.64–15; RH = 2.13). Overall, study quality was limited by high losses to follow-up (>20% in 6 studies) and short duration. Few studies adjusted for adiposity, an established diabetes risk factor.Conclusion: FBG, OGTT 2-hour blood glucose, and OGTT glucose AUC appeared to be strong and consistent predictors of subsequent T2DM among women who met diagnostic criteria for GDM using the OGTT.     

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.