Aspen En Vivo
Prometeo Archuleta
7093 Teklad shredded aspen bedding is made from 100% Great Lakes aspen wood. It makes an excellent contact bedding for rats, mice, hamsters, gerbils and guinea pigs. 7093 Shredded aspen is not a by-product of some other manufacturing process. It is manufactured from pure aspen wood, specifically for use as a contact bedding, from aged virgin timber.
7093 Shredded aspen is a natural hardwood material, so it is a preferred alternative to softwood bedding materials such as pine. Also, unlike traditional hardwood chips, the random particle sizes of shredded aspen material give animals the ability to construct nests and burrows. This makes 7093 shredded aspen a good choice for use with breeding animals. In fact, 7093 shredded aspen is the bedding material that is most widely used in Inotiv animal production barriers.
During production of 7093 shredded aspen, the product is exposed to temperatures of 1200 degrees fahrenheit for several minutes. These conditions reduce the moisture content of the finished product to approximately eight percent, and reduce harmful bacteria and other microorganisms. Laboratory analysis of the finished product is performed quarterly, and provides information on potential contaminants such as pesticides, heavy metals, yeast and molds. The QC test results are available upon request.
GV, LYFE Capital and Revelation Partners co-led the investment round with participation from additional new investors Newton Investment Management, Singapore-based global investor EDBI, LifeForce Capital, Medical Excellence Capital Partners, Mirae Asset Capital, NS Investment and others. As part of the financing, Doug Fisher of Revelation Partners will join the board of directors.
A leading iPSC platform company, Aspen combines stem cell biology with the latest artificial intelligence and genomic approaches to investigate patient-specific, restorative treatments. The company has developed a best-in-class platform to create and characterize pluripotent-derived cell medicines, which includes in-house bioinformatics, manufacturing and QC. For more information and important updates, please visit aspenneuroscience.com.
Stuart D. Kinsella, MD, MSTR, a native of Dover, Massachusetts, obtained his undergraduate degree in Biology from Harvard University, where he was a John Harvard Scholar. He obtained his medical degree from the University of Pennsylvania, and received the Mark Levine, M.D. Radiology Research Award for his work on shoulder imaging. While in medical school, he also obtained a Master of Science in Translational Research, utilizing tissue engineering techniques to develop an in vivo porcine model of bone and cartilage healing. He completed his internship and residency in the Harvard Combined Orthopaedic Residency Program, where his primary clinical and research interests shifted from sports medicine to spine. He remained in Boston at Harvard for his fellowship with the Beth Israel Deaconess Medical Center/Harvard Medical School Combined Spine Fellowship.
Dr. Kinsella began his career developing the first Globus ExcelsiusGPS robotic spine program on the east coast of Florida and performed over 100 robotic spine surgeries in just two years. He is skilled in complex open, minimally invasive, and robotic/navigation-assisted spine surgery. He has authored many clinical and translational research articles, book chapters, and presentations, and looks forward to further innovation with Steadman Philippon Research Institute (SPRI).
The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.
We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.
The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.
Transient transformation techniques are available for rapid in vivo analyses of gene function such as protein subcellular localization, protein-protein interaction, and promoter activity. For in vivo functional analyses, reporter genes such as green fluorescence protein (GFP), variants of GFP, and firefly luciferase (LUC) are common tools for molecular and cell biology studies. Protein subcellular localization, which is crucial for elucidating the cellular functions of proteins, is easily monitored by a transient expression of fluorescent fusion protein [18]. In this assay, a reporter construct harboring the gene of interest is fused with GFP or its variants and is transiently transformed into plant cells where intracellular localization is visualized through fluorescence of the reporter gene. Fluorescent proteins are also used for in vivo protein-protein interactions. Interaction assays, such as bimolecular fluorescence complementation (BiFC) and fluorescence resonance energy transfer (FRET), allow visualization of protein-protein interaction and subcellular localization of target proteins [19]. In these assays, transient co-transformation techniques with two different constructs are a convenient and practical alternative to generation of double-transformed transgenic plant and allow testing of several constructs and combinations. In addition to fluorescent proteins, LUC is used as a reporter gene mainly for measuring transcriptional activity. The LUC reporter is suitable for real-time monitoring of gene expression as it has a relatively short half-life compared to fluorescent proteins. For example, expression patterns of circadian clock-related genes, most of which show rhythmic expression in a day, are extensively examined by LUC reporter assays [20]. Typically, these assays use bioluminescence to visualize diurnal or circadian rhythms that express the LUC gene driven by a clock promoter of transgenic plants. Although studies regarding the plant clock system have principally been conducted using stable transformants, published studies report successful transient LUC assays [21, 22].
Transient transformation was monitored by the expression binary vector pPZP221-CaMV35S::EmGFP in the A. tumefaciens strain GV3101 (pMP90). Agrobacterium cells grown until stationary phase were re-suspended in an infiltration medium. Vacuum infiltration was performed using three- to four-week-old aspen cuttings without removing any tissues. When using a shoot detached from root tissues, the infiltration medium could not be evaporated from the intercellular spaces of the leaves and few cells were successfully transformed (data not shown). After three days of the transformation, GFP signals were principally observed in younger leaves and scattered in individual cells (Figure 1A-F). Most of the transformed cells were in the middle to tip region of the leaves. Although the intact plants were submerged and vacuum-infiltrated in the bacterial solution, few cells had GFP signals in petioles, stipules, stems, and roots (data not shown). GFP fluorescence was detected in various cell types such as epidermal cells, guard cells, and mesophyll cells in the leaf tissues (Figure 1G-O). This result indicates that the Agrobacterium-mediated vacuum infiltration technique can be used to analyze cells in young leaves. The validity of the tissue-dependent transformation is also described in Agrobacterium-mediated infiltration assay of Arabidopsis, where young cotyledons are more highly transformed than petioles and roots [15]. In the following studies, we principally used younger leaves for monitoring transformed cells.
We optimized the transformation conditions such as the infiltration medium, the concentration of Silwet L-77, the growth phase of bacteria, and the density of bacteria. Studies using other plant species report that these conditions influence the transient transformation efficiency in Agrobacterium infiltration technique [7, 15, 17, 25, 26]. We first estimated the effect of an infiltration medium on transformation efficiency. Two media were tested: (i) 10 mM MgCl2 and 5 mM MES-KOH (pH 5.6) established in an Agrobacterium-mediated vacuum infiltration of Arabidopsis[15] and (ii) 0.5 MS medium and 5 mM MES-KOH (pH 5.6) based on the culture medium of hybrid aspen. A saturated overnight culture of Agrobacterium was re-suspended in the media containing 200 μM Acetosyringon and then diluted to a final OD600 of 0.5. Next, 0.0075% Silwet L-77 was added to the solution before applying vacuum. Three days after the vacuum-infiltration, GFP positive cells in the younger leaves were scanned and counted in small compartments (1.5-mm2 leaf area) to evaluate the transformation efficiency. The leaves transformed in the MS solution possessed 14.6 1.2 (average SE) GFP positive cells per compartment (Figure 2A). However, fewer cells were transformed in the magnesium chloride solution (5.4 0.6 cells per compartment). Hence, we decided to use the MS medium for further estimations of transient transformation.
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