Relationship between surface marker expression and encephalitogenic potency of BP-cultured lymphocytes

The relationship between surface marker expression and encephalitogenicity of lymphocytes from various lymphoid organs of Lewis rats was studied. The encephalitogenicities after culture with BP were spleen cells greater than lymph node cells much greater than thymus cells, in this descending order. The cells from every lymphoid organ proliferated significantly in response to BP. In spleen and lymph node cells, the expression of W3/25 and OX-3 molecules on T cells increased markedly after culture with BP, but the expression of OX-19 or OX-8 molecules did not change significantly. The up-regulations of W3/25 and OX-3 molecules were more pronounced in spleen cells than in lymph node cells. Thymus cells also showed a significant increase in the W3/25 molecule after the culture with BP. Therefore, T cells from all the lymphoid organs showed a selective up-regulation of the W3/25 molecule after culture with BP, and the degree of the up-regulation seems to correspond to the encephalitogenic potency in vivo. Since the W3/25 molecule apparently plays a direct role in the effector phase of experimental allergic encephalomyelitis (EAE) by enhancing BP-reactive T cell/antigen-presenting cell interaction in the central nervous system, the up-regulation on BP-cultured T cells may strengthen interaction with the class II major histocompatibility complex molecule on antigen-presenting cells, and therefore, contribute to the efficient transfer of EAE.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

Transactivation of protein expression by rice HSP101 in planta and using Hsp101 as a selection marker for transformation

Plant HSP101 has dual activities, first, in conferring thermotolerance, and secondly, in serving as a translational activator. In this study, we introduced Oryza sativa Hsp101 (osHsp101) cDNA into tobacco by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the tobacco genome was demonstrated by Southern and Western blot analysis. Overexpression of osHSP101 had no noticeable effect on growth or development of the transgenic plants. Homozygous T(2) transgenic plants with overexpressed osHSP101 survived heat treatment better than untransformed control plants. In addition, taking advantage of conferring basal thermotolerance by plant HSP101, we were able to demonstrate the feasibility of using osHsp101 as a selection marker and select the transformants under high temperature in tobacco leaf disc transformation mediated by Agrobacterium. Furthermore, transgenic tobacco plants with overexpressed osHSP101 were able to enhance luciferase expression up to 2.9-fold more than untransformed plants in the progeny of reciprocally crossed with omega-luciferase reporter lines.     

Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells

Background  

Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process.     

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells

Aim

Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs.

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44

We have developed a systematic and generic way to improve recombinant protein productivities in stable transfections by applying mRNA and protein destabilizing elements to reduce selection marker expression strength. Interferon-gamma (IFNgamma) expression vectors containing different combinations of AU-rich elements (ARE) and mouse ornithine decarboxylase (MODC) PEST region on the amplifiable dihydrofolate reductase (dhfr) selection marker were stably transfected into CHO-DG44 cells. Improvements in specific IFNgamma productivities were 1.7-, 6.6- and 13.3-fold with the application of ARE, MODC PEST, and both ARE and MODC PEST, respectively. To further enhance productivities, compatibility of the destabilizing sequences with methotrexate (MTX) amplification was validated by amplifying the transfected cells to 50nM MTX. A 14- to 27-fold increase in specific IFNgamma productivities were observed after amplification, indicating the compatibility of the two systems. A high specific IFNgamma productivity of 1.05pg/cell/day was also attained by the amplified cell pool with both ARE and MODC PEST.     

Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability.     

Application of GFAT as a novel selection marker to mediate gene expression

The enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT), also known as glucosamine synthase (GlmS), catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. For the first time, the GFAT gene was proven to possess a function as an effective selection marker for genetically modified (GM) microorganisms. This was shown by construction and analysis of two GFAT deficient strains, E. coli ΔglmS and S. pombe Δgfa1, and the ability of the GFAT encoding gene to mediate plasmid selection. The gfa1 gene of the fission yeast Schizosaccharomyces pombe was deleted by KanMX6-mediated gene disruption and the Cre-loxP marker removal system, and the glmS gene of Escherichia coli was deleted by using λ-Red mediated recombinase system. Both E. coli ΔglmS and S. pombe Δgfa1 could not grow normally in the media without addition of glucosamine. However, the deficiency was complemented by transforming the plasmids that expressed GFAT genes. The xylanase encoding gene, xynA2 from Thermomyces lanuginosus was successfully expressed and secreted by using GFAT as selection marker in S. pombe. Optimal glucosamine concentration for E. coli ΔglmS and S. pombe Δgfa1 growth was determined respectively. These findings provide an effective technique for the construction of GM bacteria without an antibiotic resistant marker, and the construction of GM yeasts to be applied to complex media.