Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.     

Application of anoxia with glucose addition for the enhanced production of hCTLA4Ig in transgenic rice suspension cell cultures

To enhance the production of hCTLA4Ig in transgenic rice suspension cell cultures, anoxic conditions were applied during the production phase. Under the anoxic conditions in sugar-depleted media, cell viability was reduced rapidly and protease activity increased compared to aerobic conditions. However, the maximum production level of hCTLA4Ig with sugar-depleted anoxic conditions was the same as that in aerobic conditions. In addition, the production of hCTLA4Ig under anoxic conditions reached a peak 2 days earlier than that in aerobic conditions. Addition of 30 mM glucose at the production phase under anoxic conditions markedly improved cell viability. A viability level over 65% could be maintained for more than 30 days. Repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxic conditions with 30 mM glucose. In addition, the production periods of hCTLA4Ig was extended up to 30 days and the maximum production level of hCTLA4Ig under anoxic conditions was 2.1-fold higher. Therefore, anoxic conditions could be used for the enhanced production of hCTLA4Ig in transgenic rice cell cultures.     

Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using two-dimensional difference gel electrophoresis

The new technology, two-dimensional difference gel electrophoresis (2D DIGE), uses fluorescent dyes to simplify the process of detecting and matching proteins between multiple gels by allowing for the separation of up to three separate protein samples within the same gel. In this study, recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4lg) was produced in transgenic rice suspension cell cultures and the intracellular proteins were analyzed by 2D DIGE. The highest level of hCTLA4Ig (25.4 mg/L) was obtained five days after induction. The intracellular proteins expressed at both the growth and induction culture stages were separated and analyzed using DeCyder software. At least 2,218 spots were detected with two-fold thresholds and 95% confidence. We found that 29 spots increased and 20 spots decreased in their intensities during the production of recombinant hCTLA4Ig. In addition, the 2D Western blot of hCTLA4Ig revealed that this fusion protein was expressed in a variety of isoforms.     

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l⁻¹ at day 10.     

Assessment of Recovery Medium for Production of hCTLA4Ig after Cryopreservation in Transgenic Rice Cells

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.     

Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L⁻¹ in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.     

Skin-specifically transgenic expression of biologically active human cytoxic T-lymphocyte associated Antigen4-Immunoglobulin (hCTLA4Ig) in mice using lentiviral vector

Xenogeneic skin, especially porcine skin, has already been used to cover large wounds in clinic practice of wound care. Our previous data showed that transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic burn wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong its survival in xenogeneic wounds for coverage. Lentiviral transgenesis provides an extremely efficient and cost-effective method to produce transgenic animals. However, tissue-targeted transgenic expression of biologically functional protein by lentiviral transgenesis is rarely reported. In this work, a recombinant lentiviral vector (LV), named FKCW in this article, was constructed by inserting a skin-specific hCTLA4Ig expression cassette consisting of keratin 14 (K14) promoter, hCTLA4Ig coding sequence and an intronic fragment. Its efficacy for transgenesis and skin-specific expression of bio-active hCTLA4Ig protein was tested using mice as models. The LV FKCW was readily to be packaged and concentrated to high titres (1.287-6.254 × 10(9) TU/ml) by conventional lentivirus package system. Using eggs collected from only five mated females having been subjected to conventional super-ovulation treatment, 8 hCTLA4Ig transgenic founder mice were generated with the concentrated FKCW vector, and transgenic founder per injected and transferred egg was 6.3%, which was nearly 9-fold higher than that for DNA micro-injection with a similar transgene construct in our previous work. The lentiviral transgenic hCTLA4Ig exhibited strictly skin-specific expression at a level comparable to or even slightly higher than that of transgenic hCTLA4Ig delivered by micro-injection in a similar cassette. Lentiviral transgenic hCTLA4Ig protein remarkably suppressed human lymphocyte proliferation in vitro to a degree comparable to that of commercially purchased purified hCTLA4Ig protein with defined activity at similar concentrations. Besides, lentiviral hCTLA4Ig transgenic mouse skin grafted into rat burn wounds exhibited remarkably extended survival compared to wild-type skin of the same strain (13.8 ± 3.8 vs. 6.8 ± 3.0 days), indicating that lentiviral transgenic hCTLA4Ig did inhibit immune rejection against xenogeneic skin graft in vivo. These results laid down the foundation to further efficiently generate transgenic pigs skin-specifically expressing bio-active hCTLA4Ig by lentiviral transgenesis, and provided a demonstration that transgenic animals with tissue-targeted expression of biologically functional protein can be efficiently produced using LV.     

Effects of silkworm hemolymph on cell viability and hCTLA4Ig production in transgenic rice cell suspension cultures

Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at 60 degrees C for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SHadded medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.     

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig^P) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig^M), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig^P and hCTLA4Ig^M had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig^P and hCTLA4Ig^M had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ, but did not suppress the expression of macrophage-derived cytokines, including TNF-α and IL-1β, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig^P is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig^M.     

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.     

Effective delivery of siRNA to transgenic rice cells for enhanced transfection using PEI-based polyplexes

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.     

Polyclonal immunoglobulin response of thymic,hepatic and splenic lymphocytes from fetal,germ-free and conventionally reared pigs to different B-cell activators

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes withNocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells. Dedicated to Professor J. Šterzl on the occasion of his 70th birthday     

皮肤组织特异性表达hCTLA4-Ig转基因小鼠品系的建立

为研究皮肤特异性高效表达hCTLA4-Ig分子对移植皮肤存活及受体免疫功能的影响,充分实现hCTLA4-Ig分子在皮肤移植中的免疫调控功能,利用K14（角蛋白14）基因启动子,构建了hCTLA4-Ig分子皮肤组织特异性表达载体,并通过受精卵原核显微注射技术制备了K14／hCTLA4-Ig转基因小鼠并建立了品系。通过RT-PCR和Northern blot分析表明,hCTLA4-Ig在转基因小鼠体内呈皮肤组织特异性高效表达;以GAPDH基因的表达量为内部参照分析表明,hCTLA4-Ig的表达水平在不同世代之间以及转基因个体的不同生命时相点之间保持相对恒定,说明皮肤组织特异性稳定表达hCTLA4-Ig的转基因小鼠品系已被建立。     

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.     

Simulated microgravity culture system for a 3-D carcinoma tissue model

An in vitro organotypic culture model is needed to understand the complexities of carcinoma tissue consisting of carcinoma cells, stromal cells, and extracellular matrices. We developed a new in vitro model of carcinoma tissue using a rotary cell culture system with four disposable vessels (RCCS-4D) that provides a simulated microgravity condition. Solid collagen gels containing human pancreatic carcinoma NOR-P1 cells and fibroblasts or minced human pancreatic carcinoma tissue were cultured under a simulated microgravity condition or a static Ig condition for seven days. NOR-P1 cultures subjected to the simulated microgravity condition showed greater numbers of mitotic, cycling (Ki-67-positive), nuclear factor-kappa B-activating cells, and a lower number of apoptotic cells than were shown by cultures subjected to the static Ig condition. In addition, human pancreatic carcinoma specimens cultured under the simulated microgravity condition maintained the heterogeneous composition and cellular activity (determined by the cycling cell ratio and mitotic index) of the original carcinoma tissue better than static culture conditions. This new 3-D rotary cell culture system with four disposal vessels may be useful for in vitro studies of complex pancreatic carcinoma tissue.     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Stimulation of Immunoglobulin Production from Human B Lymphocytes by Staphylococcus aureus: Effects of Monocytes and Con A-Induced Suppressor Cells

Significant immunoglobulin (Ig) production by human peripheral blood lymphocytes was induced in vitro by stimulating the cells with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SpA CoI). IgG, IgM, and IgA were determined by a combination of the latex fixation test and radioimmunoassay. High levels (1,000 to 5,000 μg/ml) of IgG and IgM and a lesser amount of IgA were constantly produced during 7 to 8 days of incubation with both stimulants. Ig production induced by SpA Col stimulation was independent of the presence of T cells, while Ig production induced by PWM required T cells exclusively. Depletion of monocytes in the culture caused but a slight decrease in Ig production (particularly in the case of IgG). While the addition of a small number of monocytes enhanced IgG induction by both stimulants, coculture with an excess number of monocytes inhibited Ig induction (particularly IgG) by PWM stimulation but not by SpA CoI stimulation. Marked suppression of Ig production (IgG, IgM, and IgA) was observed in cocultures with Con A-activated T cells. The phenomena of suppression were observed in both the SpA Col-stimulated and PWM-stimulated lymphocytes. These data indicate that Ig production from B cells stimulated with a polyclonal B cell activator, SpA CoI, was independent of T cells and relatively of independent of monocytes, but could be subjected to the regulation of the Con A-induced suppressor T cells.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.