A major factor limiting the transgenesis in domestic animals is the inefficiency of maintaining large numbers of recipients carrying nontransgenic foetuses. The objectives of this study were: 1. to determine the influence of green fluorescent protein (GFP) construct injection on the development of bovine embryos, 2. to identify and select the GFP positive bovine embryos, and 3. to determine the rate of mosaicism in transgenic embryos. Cattle oocytes were matured and fertilised in vitro and zygotes were microinjected with pCX-EGFP construct consisting of CMV-IE enhancer, chicken β-actin promoter, cDNA of GFP (EGFP-732 bp) and rabbit β-globin polyadenylation sequences. Embryos from control (64) and microinjected (198) groups were cultured in vitro. After 168 h of culture, morula and blastocysts were observed in 39.06% of control and in 23.23% of injected group. We obtained three GFP positive embryos (1.51% of injected zygotes and 6.52% of morulae/blastocysts). One of them was 100, second 75 and third 25% GFP positive (66.7% of mosaicism). Use of gfp gene reporter to select bovine embryos is useful method to increase transgenic offspring, because GFP marker allows to choice only transgenic embryos and transfer them to recipients.

Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.

The tripartite-motif (TRIM)14 protein, one of the TRIM family members, was shown to participate in the antiviral and antibacterial defence. Besides, it appears to play an essential role in the processes of oncogenesis. In some types of human tumour cells, TRIM14 has been shown to inhibit apoptosis, while in others-the overexpression of TRIM14 promotes apoptosis. However, whether TRIM14 mediates apoptosis in the normal cells remains unknown. In the present study, we investigated the possible participation of the human TRIM14 gene and its mutant form (620C > T) in the induction of apoptosis in the transgenic larvae loach Misgurnus fossilis L. We observed that the expression of both forms of TRIM14 gene was accompanied by the increase of the frequency of pyknotic nuclei in fish embryos compared to control groups. Accordingly, using the TUNEL assay, the enhanced apoptosis was revealed upon expression of both forms of TRIM14 gene. The transcription of proapoptotic genes (bax, tp53, and casp9) was significantly increased in transgenic loaches expressing human wild-type TRIM14, but remained unchanged upon expression of its mutant form. In addition, the transcription of c-myc was upregulated in transgenic loaches expressing both forms. Thus, it can be assumed that during embryonic development TRIM14 has a proapoptotic effect on the cells via the activation of c-myc, tp53, and bax genes. Apparently, the mutant TRIM14 directs apoptosis via c-myc by p53-independent mechanism.

Purpose: development of new cell engineering in poultry farmingMaterials and methods. The following breeds of chickens were selected for research: Russian White, Minorca, Light Brahma and Fawn Brahma in comparison with the interspecific chimera of Japanese quail and Beijing duck. All chimeras were obtained by transplantation in the latter case of primary germ cells, in other breeds of blastodermal cells into the sub-embryonic region of recipient embryos. All embryonic cells before transplantation were cultivated for two days in special culture media supplemented with fetal cow serum and antibiotics. Chimeras were identified by the presence of contrasting feathers, unusual for this breed.Results. Unlike mammals, birds have a completely different embryonic development, which requires the development of new developments in cell biology to obtain chimeric birds. There are intrabreed chimeras and interspecies, obtained from organisms of different species. As an example of an interspecific chimera, a chimera of a Japanese quail and a Peking duck with a chimeric phenotype of black feathers on the neck is presented. Primary germ cells of Japanese quail served as donors. We obtained interbreeding chimeras of light and yellow brahma, when the donors were blastodermal cells of the light brahma, and the recipients were early embryos of the fawn brahma. Another variant of chimeras were chimeras between the breeds Russian White (donor blastodermal cells) and Minorca (embryo recipient).Conclusion. The observed sharp decline in the number of bird breeds requires new approaches to their conservation and the creation of new genetic organisms. With the help of modern developments in cell biology, it has become possible to create a new strategy for obtaining chimeric birds. The use of pluripotent embryonic cells has led to the creation of interbreed and interspecies bird chimeras.

No abstract