The following student awards were presented at the 2012 World Congress on In Vitro Biology in Bellevue, Washington. Information on additional awardees at the 2012 World Congress will be presented in the next issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website (www.sivb.org) or by contacting the SIVB Business Office at (919) 562-0600, sivb@sivb.org, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at weathers@wpi.edu.
THE 2012 WILTON R. EARLE AWARD AND
2012 SIVB STUDENT TRAVEL AWARD
Sumiyo Mimura
Prospect of Neural Cells Derived from Human Pluripotent Stem Cells for Application of In Vitro Developmental Toxicity Test
A medication which a mother may have taken during her pregnancy, in some cases causes drug-induced congenital diseases. However, there is no reliable screening test that can predict its developmental toxicity. Understanding the molecular mechanisms underlying neural development would encourage the development of alternative in vitro embryo-toxicity tests and contribute to a decreased incidence of drug-induced congenital diseases. Using a growth factor defined serum- and Xeno- free hESF-FX medium, in adherent monolayer culture, we developed a protocol to promote human pluripotent stem cell differentiation into neural cell lineage. In the culture conditions, the effect of exogenous factors can be analyzed without confounding influences of undefined components. This protocol could be used to help clarify the mechanisms underlying neural development and to assist the development of in vitro alternative developmental toxicity test for drug-induced congenital disease.
Sumiyo Mimura, Department of Oral Biology & Engineering, Division of Oral Health Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. In Vitro Cellular and Developmental Biology, 48:S44, 2012
2012 SIVB STUDENT TRAVEL AWARD
Carlos Hernandez-Garcia
Wound-induction of GmERF Promoters in Soybean
Promoters are the main regulators of gene expression at the transcriptional level. In soybean only a few promoters have been well studied, and wound-inducible promoters have received little to no attention. As plants show similar responses to mechanical wounding, pathogen invasion and damage from chewing insects, studies of wound-inducible promoters could provide insights to the mechanisms of regulation of stress-responsive genes. Here we studied induction of ten GmERF (Glycine max Ethylene Response Factor) genes and their promoters. Although the ERF gene family is one of the best characterized stress-responsive gene families, reports describing ERF promoters are surprisingly scarce. In this study, transcripts of GmERF genes accumulated to high levels in soybean seedlings wounded or treated with either methyl jasmonate or ethylene. Four GmERF promoters were subsequently isolated, fused with GFP,and reintroduced into soybean for expression analysis. In transgenic plants, the studied GmERF promoters directed low basal GFP expression in roots, pods, the epidermis, and vascular tissues. However, these promoters were highly inducible by wounding in cotyledons, hypocotyls and leaves. Wound-inducible expression was not detected in wounded roots, indicating that wound induction of these GmERF promoters is organ-dependant. Further deletion analysis for the GmERF3 promoter suggests complex regulation of expression. Candidate promoter regions likely responsible for induction and enhancement of gene expression were also identified using transient expression and expression in soybean hairy roots. This study increases our understanding of ERF promoter functionality and expands the toolbox of soybean wound-inducible promoters for potential use in both basic and applied research.
Carlos Hernandez-Garcia,The Ohio State University/OARDC, Department of Horticulture and Crop Science, Williams Hall, 1680 Madison Ave., Wooster, OH 44691.In Vitro Cellular and Developmental Biology, 48:S38, 2012
2012 SIVB STUDENT TRAVEL AWARD
Dinum Perera
Tissue Culture and Induced Mutation of Giant Miscanthus
Miscanthus xgiganteus (giant miscanthus; Mxg) is a bioenergy crop with the potential to produce liquid fuel from cellulosic biomass. Since Mxg is seed sterile, it can only be propagated vegetatively, often through rhizomes, making classical breeding techniques impossible for crop improvement. The purpose of this research was to optimize a tissue culture protocol for Mxg and to utilize the developed protocol for mutation induction. Immature inflorescence explants grown in a medium of 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.44 µM 6-benzylaminopurine (BAP) resulted in highest shoot regeneration rate. A medium containing only 2,4-D (9 µM) primarily produced direct shoots. Tissue cultured plants have been evaluated for somaclonal variations. Calli were subjected to mutagenesis using various concentrations (0.01%, 0.1%, 0.5%, 1%, 3%) of ethyl methanesulfonate (EMS) for 45 min. Effective mutagen concentration was optimized using a dose response curve and the potential mutants were regenerated. Potential mutants will be screened and evaluated in the field for mutations and will be further analyzed for genetic variability in the lab. Optimization of culture conditions, including PGR combination and concentration resulted in efficient in vitro proliferation of Mxg. The optimized protocol was helpful in generating potential Mxg mutants.
Dinum Perera, Mississippi State University, Box 9555, Mississippi State, MS 39762.In Vitro Cellular and Developmental Biology, 48:S42, 2012
2012 JOHN S. SONG AWARD
Liwen Fei
Towards Automation of Micropropagation Using a Mist Bioreactor
Micropropagation is challenging to automate mainly because of the diverse morphology of plant shoots. Previously we showed that the mist reactor can be used for 1-step propagation of carrot cells into embryos and then fully rooted plantlets using poly-L-lysine (PLL) coated polypropylene strips to which cells were attached prior to growth while hanging in the nutrient mist. Cell development into rooted plantlets occurred in 20 days. It is also possible to propagate attached small attached explants of leaves; Artemisia annua leaf explants, for example, attached by their filamentous trichomes to PLL polypropylene or nylon. Although use of an increased misting cycle led to an increased ratio in post-heart embryo formation after 14 days, continuous misting did not further increase embryogenesis. Carbon dioxide enrichment on carrot embryogenesis was also investigated. Compared with embryos developed in unvented culture chambers, after 14 days more embryos formed with 3% carbon dioxide enrichment and after 20 days the average length of rooted embryos was increased by almost 50% compared to ambient air controls. When only a sucrose solution was used during the adhesion step, cell attachment to PLL coated 50 micron nylon, 70 micron nylon and 74 micron polypropylene mesh was 4.4, 3.3 and 2.3 fold, respectively, compared to half strength B5 salts. Almost 90% of the originally attached cells remained on the 50 micron nylon mesh 24 hrs later after spraying with B5 medium in the mist reactor. Together these results demonstrate that both the mist reactor and our attachment technology may offer opportunities for at least partial automation of micropropagation.
Liwen Fei, Worcester Polytechnic Institute, 100 Institute Road, Department of Biology and Biotechnology, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 48:S76, 2012
2012 HONOR B. FELL AWARD AND
2012 SIVB STUDENT TRAVEL AWARD
Jeff Kwak
Cullin-5 Knockdown Alters Protein Expression in MDA-MB-231 Breast Cancer Cells
Breast cancer remains one of the major causes of cancer-related mortality in women in the United States. Cullin-5 (Cul5) may play a tumor suppressor role in breast cancer since the gene is located on a region of chromosome 11q22-23 that is frequently associated with loss of heterozygosity. Cul5 functions at the cellular level as an E3 ubiquitin ligase scaffold protein, and these E3 complexes target selected substrate proteins for ubiquitin-mediated degradation. To investigate the effects of decreased Cul5 expression in breast-derived cells, an in vitro Cul5 knockdown model using small interfering RNAs was established in MDA-MB-231. The efficacy of Cul5 knockdown was monitored using PCR, real-time PCR, and Western blot analysis. A non-targeting siRNA that does not target any known mammalian RNA was used as a negative control. Isolated protein from the Cul5 knockdown and the negative control samples were screened for Differential Protein Expression using 2D difference gel-electrophoresis (2D DIGE) followed by mass spectrometry to identify the differentially expressed proteins. Cul5 knockdown was confirmed at the mRNA and protein levels in the siRNA transfected cells versus the negative control transfected cells. 2D DIGE analysis identified 76 proteins that were differentially expressed in the Cul5 knockdown sample versus the negative control sample. 21 of these 76 proteins that demonstrated a 2-fold or greater differential expression were selected for further analysis by mass spectrometry. The results of the 2D DIGE and mass spectrometry for selected proteins are being validated by Western blot analysis. The identification of proteins that are differentially expressed in association with Cul5 knockdown may provide insight into the tumor suppressor functions of Cul5 in breast cancer.
Jeff Kwak, Department of Biomedical Sciences, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 48:S33, 2012
