Roland Wagner	Dieter Paul	Dirk Windgassen
Friedemann Hesse	Rainer Klocke	Christoph Priesner

Immortal, Functionally Differentiated Hepatocytes

Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This work completes a previous study using gene expression analysis (Klocke et al. 2002) of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202) consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg h-1, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP / U value was found for the three cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP / U ratio characteristic for continuous cell lines. This work demonstrates that the four transgenic and the p53-knockout hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue specific functions in immortalized cells. Christoph Priesner, Friedemann Hesse, Dirk Windgassen, Rainer Klocke, Dieter Paul, and Roland Wagner, Liver-Specific Physiology of Immortal, Functionally Differentiated Hepatocytes and of Deficient Hepatocyte-Like Variants, In Vitro Cellular and Developmental Biology - Animal, 40: 318 - 330, 2004.

Restructuring of Prostate Spheriods
Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation vs. 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55 ± 0.05 for DU 145 aggregates with projected areas exceeding 2000 m2 to a value approaching 0.75 ± 0.02 (p < 0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (p < 0.05). Combined these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production. Hong Song, Shamik K. Jain, Richard M. Enmon, and Kim C. O'Connor. Restructuring Dynamics of DU 145 and LNCaP Prostate Cancer Spheroids, In Vitro Cellular and Developmental Biology - Animal, 40:262 - 267 , 2004.

Chemodiversity of St. John's Wort Protoplasts
A procedure for protoplast isolation and plant regeneration of St. John's wort has been developed to utilize cell-to-cell variability for optimum production of valuable medicinal compounds. Calluses, induced from hypocotyl segments of St. John's wort seedlings, were used for protoplast isolation, induction ofsustained cell division and ultimately, plant regeneration. Callus-isolated protoplasts at a density of 2.0 x 105 ml-1 were embedded in 0.6 % Na-alginate blocks and cultured in a medium containing modified MS (Murashige and Skoog, 1962) salts, 2.5 µM 6-benzylaminopurine (BAP), 5.0 µM á-naphthaleneacetic acid (NAA) and 0.5 mol l-1 glucose. Protoplast-derived colonies formed compact calluses when transferred onto 0.35 % gellan gum-solidified MS medium supplemented with 2.5 µM BAP and 2.5 µM NAA. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5 µM thidiazuron (TDZ). Complete plantlets were obtained from the regenerated shoots on MS basal medium. A greater than 3-fold variation of antioxidant activity was observed among the protoplast-derived plantlets and chemically distinct germplasm lines were selected on the basis of phytochemical profiles. The protoplast to plant regeneration protocol developed in this study provides the foundation for development of novel genotypes with potential expansion of the genetic diversity through somatic hybridization, and organelle transplantation. Z. G. Pan, C. Z. Liu, S. J. Murch, and P. K. Saxena. 2004 SIVB Congress Symposium Proceedings 'Thinking Outside the Cell': Optimized Chemodiversity in Protoplast-Derived Lines of St. John's Wort (Hypericum perforatum L.), In Vitro Cellular and Developmental Biology - Plant, 41:226 - 231, 2005

Recombinase-mediated Transformation System
Current methods for creating transgenic varieties are labor and time intensive, comprising of the generation of hundreds of plants with random DNA insertions, screening for the few individuals with appropriate transgene expression and simple integration structure, and followed by a lengthy breeding process to introgress the engineered trait into cultivated varieties. Various modifications of existing methods have been proposed to speed up the different steps involved in plant transformation, as well as a few add-on technologies that seek to address issues related to biosafety or intellectual property. The problem with an assortment of independently developed improvements is that they do not integrate seamlessly into a single transformation system. This paper presents an integrated strategy for plant transformation, where the introduced DNA will be inserted precisely into the genome, the transgenic locus will be introgressed rapidly into field varieties, the extraneous transgenic DNA will be removed, the transgenic plants will be molecularly tagged, and the transgenic locus may be excised from pollen and/or seed. David Ow, 2004 SIVB Congress Symposium Proceeding: Transgene Management Via Multiple Site-Specific Recombination Systems, In Vitro Cellular and Developmental Biology - Plant, 41:213 - 219, 2005

Thinking Outside the Cell
Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continues to expand and with increasing complexity. Citrus is one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid + diploid fusion, targeted cybridization to transfer mtCMS (cytoplasmic male sterility) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement, symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and insect resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities. J.W. Grosser and F. G. Gmitter. 2004 SIVB Congress Symposium Proceedings 'Thinking Outside the Cell': Applications of Somatic Hybridization and Cybridization in Crop Improvement, with Citrus as a Model, In Vitro Cellular and Developmental Biology - Plant, 41: 220 - 225, 2005