Analysis of the transcriptional activity of model piggyBac transgenes stably integrated into different loci of the genome of CHO cells in the absence of selection pressure.

CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably integrated into the genome of CHO cells using the piggyBac transposon. Each individual transgene contained a unique tag, a DNA barcode, and the resulting polyclonal cell population was cultured for almost a month without any selection. Next, using the high-throughput sequencing, genomic localizations of barcodes, as well as their abundances in the population and transcriptional activities were identified. In total, ~640 transgenes more or less evenly distributed across all chromosomes of CHO cells were characterized. More than half of the transgenes were completely silent. The most active transgenes were identified to be inserted in gene promoters and 5' UTRs. Transgenes carrying Chinese hamster full-length promoter of the EF-1α gene showed the highest activity. Transgenes with a truncated version of the same promoter and with the mouse PGK gene promoter were on average 10 and 19 times less active, respectively. In total, combinations of genomic loci of CHO cells and transgene promoters that together provide different levels of transcriptional activity of the model reporter construct were described.

Discrepancy between thermodynamic and kinetic stabilities of the tert-butanol hydrates and its implication for obtaining pharmaceutical powders by freeze-drying.

The tert-butanol (TBA)-water system is studied in relation to increasing the efficiency of obtaining pharmaceutical powders by freeze-drying. Trehalose was used as a model target product. We report the X-ray diffraction and thermal analysis data which add surprising new information to the phase diagram of this previously repeatedly studied system. The freezing protocol has a strong impact on the specific surface area of the trehalose freeze-dried cakes and on the primary drying time. This is related to a discrepancy between the kinetic and thermodynamic stabilities of several TBA hydrates: di-hydrate (H1), heptahydrate (H2), and decahydrate (H3).

Large porous particles for respiratory drug delivery. Glycine-based formulations.

Large porous particles are becoming increasingly popular as carriers for pulmonary drug delivery with both local and systemic applications. These particles have high geometric diameters (5-30µm) but low bulk density (~0.1g/cm3 or less) such that the aerodynamic diameter remains low (1-5µm). In this study salbutamol and budesonide serve as model inhalable drugs with poor water solubility. A novel method is proposed for the production of dry powder inhaler formulations with enhanced aerosol performance (e.g. for salbutamol-glycine formulation the fine particle fraction (FPF≤4.7µm) value is 67.0±1.3%) from substances that are poorly soluble in water. To overcome the problems related to extremely poor aqueous solubility of the APIs, not individual solvents are used for spray freeze-drying of API solutions, but organic-water mixtures, which can form clathrate hydrates at low temperatures and release APIs or their complexes as fine powders, which form large porous particles after the clathrates are removed by sublimation. Zwitterionic glycine has been used as an additive to API directly in solutions prior to spray freeze-drying, in order to prevent aggregation of powders, to enhance their dispersibility and improve air-flow properties. The clathrate-forming spray freeze-drying process in the multi-component system was optimized using low-temperature powder X-ray diffraction and thermal analysis.

[Role of GAGA Factor in Drosophila Primordial Germ Cell Migration and Gonad Development].

The GAGA protein of drosophila is a factor involved in epigenetic transcription regulation of a large gene group controlling developmental processes. In this paper, the role of GAGA factor in germ cell migration is demonstrated as well as its effect on the gonad development in drosophila embryogenesis. Mutations in the Trl gene, encoding GAGA factor, prematurely induces the active migration program and relocation of the primordial cells inward the embryo before the beginning of gastrulation. The germ cells that prematurely separated from the main group migrate ectopically, lose orientation, and stay out of gonad development. Expression pattern of the Trl gene suggests its activity in epithelial cells of the embryonic blastoderm, part of which contact primordial cells. Thus, GAGA factor influences migration of these cells in an indirect manner via their somatic environment.

[Basic aspects of ovarian development in Drosophila melanogaster].

Modern views of the development and structural organization of the female reproductive system in Drosophila melanogaster are reviewed. Special emphasis is placed on the generation and development of follicles in the germarium and the interactions of germline and somatic cells in the egg chamber. Detailed consideration is given to the main events that ensure and regulate the transport of mRNA, proteins, and organelles from nurse cells to the oocyte in the germarium and at later stages of egg chamber development.

[Effect of hypomorphic mutation in Trithorax-like gene on Drosophila melanogaster oogenesis].

We generated and characterized a new hypomorphic mutation of Drosophila melanogaster Trithorax-like (Trl) gene named Trl362. The Trl362 homozygous females are sterile and lay a small number of eggs; most embryos die at the early developmental stages. The transcriptional Trl level of adult Trl362 females was markedly lowered. Little or no GAGA protein, encoded by Trl@, was detected in the nurse cell nuclei. The ovaries of Trl362 females showed impairments, such considerable changes in the structure of both ovarioles and individual egg chambers. We believe that the observed ovarian defects in Trl362 mutants are mostly due to a decreased amount of GAGA protein in the germline cells. An increase of GAGA-519 protein caused by introduction of hsp83:GAGA-519 transgene against Trl362 background rescued partially the female fertility. It may well be that a decrease of GAGA protein in Trl362 germline cells leads to a defective expression of the genes regulated by transcription factor GAGA, whose products are essential for normal Drosophila oogenesis.

[Generation and analysis of novel mutations of the trithorax-like gene in Drosophila melanogaster].

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional GAGA factor. The expression of Trl is known to depend on numerous factors, such as the organ, the tissue, the ontogenetic stage, and the ambient temperature. Apparently, this expression is controlled by a complex system of regulatory elements, which so far has been scarcely studied. Our preliminary results indicate that the second intron of the Trl gene bears functionally significant elements. To test this assumption, we generated 23 novel alleles of the gene via P-induced male recombination and analyzed them cytogenetically. Of these mutations, 13 (recessive lethals) are deletions, disrupting the coding gene region. Ten mutations (seven deletions and three duplications) remove parts of the second Trl intron only. Some of these mutant stocks exhibit lower viability at different temperatures. These results suggest that the second intron region harbors functionally significant elements. The deletion mapping results verified the localization of the Trl gene in the 70F1-2 region.