Characteristics of Mel Ibr Melanoma Line Subclone after Treatment with Chicken Embryo Extract.

We continue analysis of the phenotype of human melanoma cell Mel Ibr subclone obtained previously by treatment of the parental cell line by chicken embryo extract. The present study is focused on detection of markers of epithelial-mesenchymal transition that determine enhanced metastatic and invasive potential of malignant tumors of various locations. Analysis of the expression of E-cadherin and vimentin genes in the subclone and parental cells detected activation of epithelial-mesenchymal transition in the subclone. Immunological markers CD90, CD271, and CD95 were present in the parental population, but were not detected on the subclone cells. In contrast to the parental line, cells of the analyzed subclone retain viability in serum-free medium and formed vessel-like structures characteristic of vasculogenic mimicry.

Changes in the Morphological and Immunological Characteristics of Mel Ibr Melanoma Cells in Response to Chicken Embryo Extract.

Exposure of Mel Ibr human melanoma cells to chicken embryo extract resulted in the appearance of a subclone with morphological and growth characteristics similar to those of embryonic stem cells. The subclone differed from the parental line cells by a sharply reduced percentage of HLA-DR(+) and CD54(+) cells, a significantly elevated percentage of CD63(+) cells, and appearance of CD133(+) and Oct-4A(+) cells. Hence, the subclone cells were characterized by the same features as stem tumor cells and could be responsible for further progress of tumor growth.

[Development of transgenic chicken with a gene of human granulocyte colony-stimulating factor using sperm-mediated gene transfer].

Transgenic poultry with a gene of human granulocyte colony-stimulating factor (gcsf) was developed by arti- ficial insemination with the transfected sperm using the "Lipofectamin 2000" reagent. The ratio of trans- genic chickens carrying the human gcsf genome was 33.3% of the total number of birds obtained. It was shown that the concentration-of the human G-csfprotein in blood serum oftransgenic specimens varied from 50 to 220 pg/ml. The first generation of poultry was obtained as a result of insemination oftransgenic hens by sperm of anti-transgenic cocks, thereby inheritance of the foreign gene was found in 37.5% of the obtained chickens.

[Efficacy of various methods of a reporter gene transfer to chicken embryonic cells].

The methods of transfection ofa plasmid with a reporter gene involving DNA injection into chicken embryonic cells were studied. The parameters of the efficient transfection of chicken blastodermal cells with a foreign gene have been determined (20-24 and up to 40% in culture and embryos, respectively). A high efficiency of transfection of primordial germ cells isolated from the gonads has been obtained after DNA injection into the dorsal aorta of 2.5-day-old chicken embryos.

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients.

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.