A high repetition rate (80 MHz) picosecond pulse (approximately 2 psec) infrared laser was used for the inactivation (functional enucleation) of oocytes and two-cell mouse embryos and also for the fusion of blastomeres of two-cell mouse embryos. The laser inactivation of both blastomeres of two-cell mouse embryos by irradiation of nucleoli completely blocked further development of the embryo. The inactivation of one blastomere, however, did not affect the ability of the second intact blastomere to develop into a blastocyst after treatment. Laser inactivation of oocytes at Metaphase II (MII) stage and parthenogenetically activated pronuclear oocytes also completely blocked their ability for further development. Suitable doses of irradiation in cytoplasm region did not affect the ability of embryos and activated oocytes to development. The efficiency of laser induced fusion for blastomeres of two-cell embryos was 66.7% and all the tetraploid embryos developed successfully into blastocysts in culture. Our results demonstrate unique opportunities of the applications of a suitable infrared periodic pulse laser as a universal microsurgery tool for individual living cells.
Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar. The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition. It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes. The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence. We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.
Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.
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