A new micromanipulation technique permitted the scrambling of the zygote cytoplasm. Such interference had no effect on preimplantation development, and when zygotes with scrambled cytoplasm were transfered to the pseudopregnant females, normal and fertile mice were born. This demonstrates that no morphogenetic factors are prelocalized in the egg cytoplasm. Cleavage characteristics of mouse embryos provide the evidence that zygote cytoplasm does not define any determinate type of cleavage. We conclude that the mechanism of ooplasmic segregation is not used in the mouse (and presumably mammalian) development. It is suggested that the turning point in the evolution of mammalian embryogenesis was the transition to the intrauterine development, that started the process leading among other changes, to the loss of the ooplasmic morphogenetic determinants.
Low viability of manipulated or in vitro cultured embryos is caused primarily by the reduced cell number in the implanting blastocysts. In order to investigate the effect of implantation delay on embryo viability and cell number, mouse blastocysts were transferred into oviducts of day 0 pseudopregnant females. This type of transfer improved embryo survival rates, indicating that embryos retarded by in vitro culture restored their viability during 3 days of delayed implantation. Our results showed that even in the cases when the initial cell count was as low as 28.2 0.7 cells per blastocyst (vs. 60.5 f 1.4 cells in the control blastocysts, developed in vivo), implantation delay increased this number to 107.2 2 3.5 cells (control blastocysts had at this stage on average 111.0 3.7 cells). Half-blastocysts, developed from the single blastomeres of the 2-cell embryos or from experimentally produced tetraploids, had around 50 cells after 3 days of implantation delay. This indicates that the start of blastocyst dormancy is triggered during the eighth cell cycle and independent of the absolute cell number or the number of cytokineses. Implantation-delayed blastocysts, developed from the half-embryos with the doubled volume of cytoplasm, had on average 70.5 * 2.4 cells, suggesting that embryo fall into quiescence is also dependent upon the attainment of a definite nucleo-cytoplasmic ratio. We conclude that blastocyst readiness for implantation is determined by two factors: number of cell cycles and nucleo-cytoplasmic ratio.
A physical map of the M. neustria nuclear polyidrosis virus (ManeNPV) genome was constructed, the complete order of BamHJ, Kpnl and Pstl restriction enzyme sites was determined t a polyhedrin gene was localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclease profiles of the DNA of ManeNPV plaques isolate propagated in A. pernyi cells demonstrated ^persistent heterogeneity*, submolar bands were shown to appear in a digestion pattern of DNA of the first passage virus. These bands were proved to be due to the DNA molecules presence in the non-homogeneous virus DNA pool, their chains having been shown to carry a putative break in a definite site. Such a *break site» was localized on the physical, map of ManeNPV genome. The Baculoviridae contain double-stranded circular DNA viruses infecting a lot of species belonging mainly to the Lepidoptera> Diptera> and Hymenoptera orders. Baculoviruses with their large genomes and complex reproduction cycle accompanied by the cas cade gene expression regulation have become an interesting and well-made topic of molecular biology studies. The interest to the representatives of this family is also caused by three aspects of their practical use. First of them is an application of the baculoviruses as vectors for gene therapy, the problem being investigated during last decade [1, 2]. Two other aspects are more traditional: baculoviruses are considered as viral insecticides for pest insect control and are widely used as vectors for recombinant protein synthesis in both in vivo and in vitro cul tivated insect cells [3 J. M. neustria {Mane) larvae (Lepidoptera, Lasiocampidae) is a well known garden fruit pest insect, especially on apple trees. However, outbreaks of the pest mass reproduction occur often also in woods (first of all, in oak woods) of the forest-steppe zone.
The recombinant baculovirus vector with EGFP reporter gene under the control of strong CAG promoter cassette was used for transduction of normal (HEK293) and tumor (HeLa) human cell lines. Dependence of transduction efficiency on the virus dose, time of virus incubation, temperature and cell type is shown. Transient reporter gene expression gradually diminished and was detected in single cells on the 15 th day after transduction.
In this review the literature data are analyzed relative to the study of a new vector system for the cells of vertebrates, based on the insect viruses – baculoviruses. The ways and mechanisms of recombinant baculoviruses penetration into cells, the factors, which influence the effectiveness of transduction, the principles of the modification of virus display, and the reaction of the different types of cells on virus introduction are examined. The prospects of using recombinant baculoviruses in cellular engineering are discussed
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