2004 WILTON R. EARLE AWARD and 2004 SIVB STUDENT TRAVEL AWARD

Transgene Expression in Site-specific Integrant Rice Lines

Site-specific gene integration method based on heterologous system such as cre-lox has the potential to stabilize transgene expression. The molecular strategy allows the production of site-specific integration locus but it doesn't prevent random integrations. Therefore two types of integrant lines are produced single-copy and multi-copy. The objectives are (1) to test stability of transgene expression, (2) to determine whether random integrations are linked to the target locus in transformed plants. Consistent transgene expression (gus) was observed in single-copy lines, whereas high variability observed in multi-copy lines. Progeny analysis of single-copy lines was carried on three lines, which reveal stable inheritance of the locus and a positive gene dosage effect i.e. homozygous plants contained twice as much expression level as hemizygous plants. Molecular and genetic data will be presented to demonstrate consistent and stable transgene expression in site-specific integrant lines. Molecular detail of the strategy will also be presented.

Magnolia Ariza-Nieto, University of Arkansas Crop Soil and Environmental Sciences, 115 PNSC, Fayetteville, AR 72703. In Vitro Cellular and Developmental Biology, 40: 29-A, 2004.

2004 HOPE E. HOPPS AWARD and 2004 SIVB STUDENT TRAVEL AWARD

Hypothermic Storage of Neonatal Mammalian Cardiomyocytes: Assessment of Multiple Markers of Viability

Hypothermic storage of primary cells is emerging as the rate-limiting step to the application of Regenerative Medicine. Despite many attempts, successful extension of the cold storage interval of myocytes has not been realized. In this study, an evaluation of several hypothermic storage solutions in the neonatal rat ventricular cardiomyocyte model (NRVCM) was performed. Samples were assessed by a panel of indicators that measured membrane integrity, metabolic activity and spontaneous contractile function. To discern differences in the various solutions 2',7'-dichlorofluorescein and western blotting analysis were performed. Cultured NRVCM were held at 4C for 24-72 hrs in either standard culture media, ViaSpan® (Univ. of Wisconsin) or HypoThermosol® (HTS) variants (HTS-Base, HTS-DCC or HTS-FRS). Samples stored in HTS-DCC and HTS-FRS yielded overall survival rates 10-30% greater than the base solution (p<0.05), and >50% versus cells stored in conventional media (p<0.01). Upon return to normothermic conditions, NRVCM stored in HTS-FRS for 24-48hrs regained spontaneous contractions and 90-95% metabolic activity as compared to 37C controls. Extending the storage time to 72hrs resulted in extensive cell loss in all solutions, except cells stored in HTS-FRS, which maintained 50% viability and function post-storage. Western blot analysis revealed that extended/suboptimal storage conditions resulted in a 5-fold increase in AIF protein levels in adherent cells following storage. Successful preservation of NRVCM for 48hrs is possible in cold storage solutions that provide protection from the cellular and molecular stress encountered during and following hypothermic storage. These results provide a foundation towards increasing the cold storage window for sensitive biologic products.

Kristi K. Snyder, Binghamton University, Biological Sciences, Science 3, Rm. 210, Binghamton, NY 13902-6000. In Vitro Cellular and Developmental Biology, 40: 37-A, 2004.

2004 JOHN S. SONG AWARD
A Barley LTP Promoter for Tissue-specific Expression of Transgene-mediated Disease Resistance

Transgene-mediated disease resistance is a promising means for controlling crop diseases such as Fusarium Head Blight (FHB). A successful strategy should combine the use of effective pathogenesis-related (PR), disease resistance cascade regulatory genes, or RNAi constructs under appropriate tissue-specific promoters. We have cloned and characterized a novel barley gene, Ltp6, which is highly expressed in the pericarp epidermis, one of the first spike tissues to be colonized by Fusarium graminearum, the main causal pathogen of FHB. The open reading frame encoded a polypeptide of 124 amino acids showing 87% identity with WBP1A, a wheat lipid transfer protein (LTP), but much lower homology to other barley LTPs. Expression analyses showed that this LTP gene is also highly expressed in the coleoptile and embryo, but not in leaves, stems, roots or other spike tissues. Low expression was found in the ovary. In addition, Ltp6 mRNA levels increased during ABA, SA, NaCl and cold treatments in seedling tissues. Taken together, the tissue-specific and response patterns of Ltp6 were distinct from other known barley LTPs. Series of Ltp6promoter deletions were studied in transient expression assays using sgfpas a reporter. Quantitative RT PCR was used to assess the level of transcription conferred by the different promoter constructs. All constructs containing at least 191 bp of upstream sequence, and the 5'UTR, retained most of the promoter activity. Deletion of a 64 bp fragment (-191/-127) resulted in an 80% drop in expression. The suitability of this promoter for engineering transgene-mediated resistance to FHB and other diseases will be discussed with respect to spatial, temporal and inducible patterns of expression of the native and reporter genes in control and transgenic barley plants.

Maria L. Federico, University of Wisconsin-Madison, Agronomy Department, 1575 Linden Drive, Madison, WI 53706. In Vitro Cellular and Developmental Biology, 40: 37-A, 2004.

2004 PHILIP R. WHITE AWARD

Dorothy M. Gillespie - Nashua High School, New Hampshire. Dorothy Gillespie, a biology teacher at Nashua High School in Nashua, New Hampshire, is the recipient of the 2004 Philip White Memorial Award. Ms. Gillespie would like to introduce plant tissue culture next year in her Advanced Placement Biology course. She will use the White Funds to attend a workshop entitled "2004 NH Biotechnology Institute" at the Seacoast School of Technology, in New Hampshire in June and will purchase supplies for her classroom. Over the summer months Dorothy will be developing experiments and lesson plans for her classroom with the help of donated supplies from Caisson Laboratories, Inc., Plant Cell Technology, Inc, and Kitchen Culture Kits, Inc. From Ms. Gillespie:

"Thank you for awarding me the Phillip White Memorial Scholarship. The money from this grant will be used to fund several tissue culture experiments in my Advanced Placement Biology class. I attended a teacher workshop on tissue culturing where we set up African violet cultures and learned sterile technique and some aspects of commercial plant culturing. This summer, I planned the labs I will set up this year. My students will first learn sterile technique and then set up African violet cultures, which will be followed through to maturity. Later they will culture carrots and seeds using different growth hormones in the media. There are many ways in which tissue culturing will demonstrate and reinforce important biological concepts. Our discussions will include microbes, nutrient uptake, cloning, somatic cell mitosis, cell signaling, differentiation, plant tissues, and environmental factors that affect growth.

At this time, I have used part of the funds to purchase a digital camera, memory card, and battery charger to take pictures of the students in lab and their research progress. Students will be able to use the camera and we will incorporate the images into PowerPoint presentations. I have also constructed three PVC clean boxes and have purchased some of the other supplies and equipment such as a microwave, alcohol, bleach, violets, potting soil, spray bottles, and knives. The rest of the supplies are still on my to do list. I am very excited about pioneering a new lab technique in my school. We have an environmental chamber for the cultures and, in a year, I hope to be able to share my experiences with other teachers. All of the funds will be used in my classroom for tissue culture. I truly appreciate this opportunity and am honored to be the recipient of the Phillip White Memorial Scholarship."

Sincerely,
Dorothy Gillespie
South Nashua High School
Nashua, NH