Uniform Expression and Relatively Small Position Effects Characterize Sister Transformants in Maize and Soybean.

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

Modification of AlphaLISA Excitation Wavelength Leads to Improved Assay Sensitivity for Photosynthetic Tissue Samples.

For the purposes of high-throughput immunoassay screening, PerkinElmer's AlphaLISA technology offers many benefits over traditional enzyme-linked immunosorbant assay (ELISA). However, its 680 nm excitation wavelength coincides with a wavelength of peak photosynthetic pigment absorbance, hindering the technology's utility within the plant biotechnology industry. In assays containing photosynthetic matrices, it is proposed that excitation of chlorophyll leads to the production of singlet oxygen, which initiates a pigment-associated background signal, reducing assay sensitivity. A customized donor bead, modified for excitation outside the range of photosystem absorbance, was tested for its capacity to improve assay sensitivity with extracts containing photosynthetic pigments. In three assays designed against crystalline domain insecticidal protein targets, use of the customized donor bead along with its altered excitation wavelength led to the elimination of pigment-associated signal and improved separation between target-positive and null samples. Reduction in null photosynthetic extract signal led to a 16× sensitivity improvement in a quantitative assay. The customized donor bead was also found to be photostable under ambient laboratory lighting, potentially improving the overall utility of AlphaLISA technology. The customized donor bead enables sensitive, high-throughput immunoassay screening of photosynthetic tissues within the plant biotechnology industry using a convenient, photostable protocol.

Labeling of bacterial pathogens for flow cytometric detection and enumeration.

Traditional uses of flow cytometry have been for research and diagnostic purposes, on mammalian cells. This unit focuses on using a flow cytometer for enumerating specific bacteria and parasites. Several different labeling methods are discussed, as well as how to set up a cytometer for detecting and enumerating bacteria. Labeling methods include direct detection with a primary fluorochrome-conjugated antibody and indirect labeling with an unconjugated primary and a fluorochrome-conjugated secondary antibody, as well as labeling with rRNA sequence-specific peptide nucleic-acid probes end-labeled with a fluorochrome. Data and methods throughout this unit focus on detection of Salmonella in clean matrices; however, pertinent information is also provided on using these methods to label other pathogens. The Commentary covers critical aspects of the protocols, and also includes information and suggestions on the application of these methods for testing in the food and pharmaceutical industries, as well as in environmental water testing.

Resistance of Lactobacillus casei in plastic-composite-support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system.

Lactic acid fermentations were performed with plastic-composite-support (PCS) disks in solvent-saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent-saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent-saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent-detoxifying properties, two bioassays were developed. When solvent-saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent-saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso-octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na(2)CO(3) stripping solution. Also, recovery by emulsion liquid extraction in the hollow-fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow-fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid-liquid extractive integrated fermentations.

Five-color flow cytometric analysis of swine lymphocytes for detection of proliferation, apoptosis, viability, and phenotype.

BACKGROUND: The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin. RESULTS: Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin (cell surface antigen), Texas Red phycoerythrin tandem (cell surface antigen), allophycocyanin (annexin V), and 7-amino-actinomysin D (7AAD; viability). For analysis, gates were set on live (annexin V(-), 7AAD(-)), intact apoptotic (annexin V(+), 7AAD(dim)), and live plus intact apoptotic (annexin V(+/-), 7AAD(dim/-)) cells, and the phenotypes of PBMCs within these populations were determined during the course of the in vitro response. Dead cells (i.e., 7AAD(bright)) were excluded from the analysis. CONCLUSION: Application of this method for the determination of porcine lymphocyte subset proliferation is presented.