Molecular cloning of protamine-2 and expression with aging in Japanese monkey (<Emphasis Type="Italic">Macaca fuscata</Emphasis>)

In order to isolate genes relating spermatogenesis with postnatal development and aging, we have attempted to obtain genes showing differences in expression in testis of Japanese monkeys (Macaca fuscata) by means of differential display PCR, and we have cloned, sequenced and characterized protamine-2 (PRM2) of Japanese monkey. The predicted open reading frame encoded a protein of 103 amino acid residues, most of which are common to those of other mammals. Northern analysis revealed that the PRM2 gene is expressed at adult and aged stages, but not at the juvenile stage. In situ hybridization revealed that the PRM2 gene is expressed mainly in late spermatids and its expressional potential is decreased from adult to aged stages. It suggests that PRM2 in spermiogenesis is mediated by the age of the animal.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

Improved recombinant gene expression in CHO cells using matrix attachment regions

The use of animal cells such as Chinese hamster ovary (CHO) cells for recombinant gene expression provides many advantageous features such as proper folding and post-translational modification of the recombinant protein. However, recombinant genes introduced into animal cells are often expressed at low levels mainly due to position effects from the neighboring chromatin context. The tedious and time-consuming selection and amplification procedure has been the major hurdle for using animal cell line such as CHO cells. To improve mammalian cell expression systems, we screened a variety of matrix/scaffold attachment region (MAR/SAR) elements for their ability to insulate transgene expression from the position effects in CHO cells. We found that the human beta-globin MAR element is particularly effective as the frequency of beta-Gal positive colonies was increased by up to 80%. The expression levels of these colonies were also enhanced about seven-fold. These improvements appear to be related to the increased copy numbers and a higher efficiency of expression of the integrated genes. When this element was used to express soluble TGF-beta type II receptor (sTbetaRII) through the gene amplification system, the frequency of colonies expressing detectable amounts of sTbetaRII was much higher than that of the control vector. We could also generate high sTbetaRII producers with uniform growth properties by a simple two-step amplification process involving two concentrations of methotrexate. This eliminates the need to isolate individual colonies followed by multi-step treatments of methotrexate and thereby greatly simplifies this mammalian expression system.     

Synthesis of oligodeoxynucleotides containing a single diastereoisomer of alpha-(N2-2'-deoxyguanosinyl)-N-desmethyltamoxifen

A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was described. The site-specifically modified oligodeoxynucleotide can be used for mutagenesis, DNA repair, and 3D structural studies and also as standard for quantitative analysis of TAM-DNA adducts in animal and human.     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.     

G-PRIMER: greedy algorithm for selecting minimal primer set

G-PRIMER, a web-based primer design program, has been developed to compute a minimal primer set specifically annealed to all the open reading frames in a given microbial genome. This program has been successfully used in the microarray experiment for analyzing the expression of genes in the Xanthomonas campestris genome. AVAILABILITY: It is available at http://mammoth.bii.a-star.edu.sg/gprimer/. Its source code is available upon request.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.