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1.
Bioorg Khim ; 34(2): 185-93, 2008.
Article in Russian | MEDLINE | ID: mdl-18522274

ABSTRACT

A biotechnological system for the production of human beta interferon was developed on the basis of a hybrid gene constructed from the coding sequence of the beta interferon gene inserted into the first exon of the sheep beta lactoglobulin gene. It is intended for the expression of human beta interferon in mammary glands of transgenic animals. Two lines of transgenic rabbits were obtained using the hybrid gene. The tissue specificity of the expression of the transgene and the frequency of its inheritance in the first and second generations were studied. The activity of interferon was 2.2 x 10(4)-7.2 x 10(4) IU per milliliter of milk of transgenic female rabbits. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.


Subject(s)
Interferon-beta/biosynthesis , Mammary Glands, Animal/metabolism , Animals , Animals, Genetically Modified , Exons , Female , Humans , Interferon-beta/genetics , Lactoglobulins/genetics , Milk/metabolism , Milk Proteins/metabolism , Organ Specificity , Rabbits , Sheep
2.
Izv Akad Nauk Ser Biol ; (3): 284-91, 2006.
Article in Russian | MEDLINE | ID: mdl-16771141

ABSTRACT

Cloned bovine embryos were produced at the blastocyst stage. Prior to enucleation, oocytes were freed from the zona pellucida. Fibroblasts isolated from the bovine fetus were used as nuclear donors. Pairs of fetal fibroblasts and enucleated oocytes (cytoplasts) were glued in phytohemagglutinin solution under a binocular microscope. The subsequent electrofusion of 39 fetal fibroblast-cytoplast pairs yielded 36 reconstructed one-cell embryos (92.3%). After culturing in synthetic oviduct fluid for 7.5 days, seven cloned embryos developed to the blastocyst stage (19.4%) and six blastocysts were considered fit for transplantation. The applied technique of bovine embryo growth allowed 31.1% zona-free oocytes parthenogenetically activated by to reach the blastocyst stage.


Subject(s)
Blastocyst , Cloning, Organism , Fetus , Fibroblasts , Nuclear Transfer Techniques , Oocytes , Animals , Blastocyst/cytology , Cattle , Embryo Transfer , Female , Fetus/cytology , Fibroblasts/cytology , Oocytes/cytology , Zona Pellucida
3.
Ontogenez ; 33(2): 100-6, 2002.
Article in Russian | MEDLINE | ID: mdl-11969068

ABSTRACT

We studied the capacity of rabbit oocytes for electrofusion with morula blastomeres and fetal fibroblasts. The morula blastomeres fused with aging ooplasts more readily than the fetal fibroblasts: 92.9 versus 63.0%, p < 0.001. The fetal fibroblasts fused with young enucleated oocytes more efficiently than with the aging ones: 98.4 versus 63.0%, p < 0.001. Serum starvation of the fetal fibroblasts in DMEM medium for 7-14 days reduced their capacity for fusion with young ooplasts, as compared to that after starvation for 0-4 days: 67.2 versus 98.9%, p < 0.01). The increased time of "starvation" in an "impoverished" medium reduced the capacity of fetal fibroblasts with aging ooplasts as compared to the fibroblasts cultivated in the full medium and in the "impoverished" medium for one or two days: 64.5 versus 37.4%, p < 0.01. Hence, the efficiency of the fusion of the oocytes with nuclear donor cells depends on the age of the recipient oocyte, the origin of nuclear donor cells, and the conditions of cultivation.


Subject(s)
Blastomeres , Cell Fusion/methods , Cell Nucleus , Oocytes/physiology , Animals , Cells, Cultured , Electrophysiology/methods , Female , Fibroblasts , Rabbits
4.
Ontogenez ; 32(2): 130-9, 2001.
Article in Russian | MEDLINE | ID: mdl-11544764

ABSTRACT

We studied the capacity of nuclei of rabbit fibroblasts taken from various developmental stages for reprogramming in the cytoplasm of mature aging enucleated oocytes and development of the cloned embryos to the preimplantation stages. A negative correlation was found between the age of an animal--donor of fibroblasts and efficiency of the development of cloned embryos (rmorula-blastocyst = -0.826, rblastocyst = -0.7139). A reliably decreased capacity for reprogramming of the nuclei of donor fibroblasts was shown upon transition from prenatal development to the postnatal one, as well as a trend to a decreased capacity of nuclei for reprogramming during aging. Aging of cells in the culture, at least until the 10th passage, did not affect the capacity of the nuclei of fetal fibroblasts for reprogramming and development of cloned embryos.


Subject(s)
Age Factors , Cloning, Organism , Embryonic and Fetal Development , Nuclear Transfer Techniques , Animals , Cells, Cultured , Cellular Senescence , Female , Rabbits
5.
Biochemistry (Mosc) ; 66(4): 378-83, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11403643

ABSTRACT

Technology for preparation of chymosin from milk of transgenic sheep has been elaborated. Purification of the preparation by ion-exchange chromatography on aminosilochrom and biospecific chromatography on bacitracin-Sepharose yielded homogeneous active enzyme. Hydrolysis of protein substrates (hemoglobin, BSA, and sodium caseinate) by the transgenic sheep chymosin and stability of the enzyme at various values of pH were studied. Judging by the amino acid composition, the N-terminal sequence involving six amino acid residues, molecular mass, stability at various pH values, and the catalytic activity against the protein substrates, the transgenic sheep chymosin is identical to calf chymosin.


Subject(s)
Aspartic Acid Endopeptidases/isolation & purification , Chymosin/isolation & purification , Milk/enzymology , Amino Acids/chemistry , Animals , Animals, Genetically Modified , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Cattle , Chromatography, Ion Exchange/methods , Chymosin/chemistry , Chymosin/metabolism , Electrophoresis/methods , Enzyme Activation/physiology , Enzyme Stability/physiology , Hydrogen-Ion Concentration , Sheep , Species Specificity , Substrate Specificity/physiology
6.
Ontogenez ; 30(6): 411-6, 1999.
Article in Russian | MEDLINE | ID: mdl-10624714

ABSTRACT

Successful transplantation of mammalian nuclei from differentiated cells has become possible after the application of original methods directed at the synchronization of cell cycles of the donor cell and recipient cytoplasm. We obtained a line of rabbit fetal fibroblasts which was used to study factors affecting the success of reprogramming. The nuclei of fetal fibroblasts (up to the 10th passage inclusive) proved to be capable of reprogramming and ensuring development of the cloned embryos until the preimplantation stages. The influence of synchronization of the cell cycles of the nucleus donor and recipient on the efficiency of reprogramming was studied. The rate of development of the cloned rabbit embryos to the morula-blastocyst stage reached 67% when the nuclei used were from stationary culture cells (G0-phase).


Subject(s)
Cell Nucleus/drug effects , Culture Media, Serum-Free/pharmacology , Fibroblasts/drug effects , Animals , Cell Culture Techniques/methods , Cell Cycle , Cell Differentiation , Cell Nucleus/ultrastructure , Cell Separation/methods , Cells, Cultured , Embryo, Mammalian , Female , Fibroblasts/ultrastructure , Nuclear Transfer Techniques , Pregnancy , Rabbits
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