EFFICIENCY OF LOCAL TRANSGENESIS OF THE OVIDUCTAL CELLS IN CHICKEN AS INFLUENCED BY HORMONAL STIMULATION

Abstract

A b s t r a c t One of the promising areas of biotechnology is to create transgenic chicken bioreactors. However, despite notable successes in avian transgenesis, the creation of transgenic chickens now is a particular problem. Searches and development of alternative methods of directed gene transfer are required, one of which is genetic transformation of certain organs, in particular chicken oviduct (somatic transgenesis). This can significantly reduce the expenditure of time and materials in the preparation of transgenic organisms than using other cellular targets for targeted delivery of DNA (cell blastoderm, primordial germ cells, embryonic stem cells) because the genetically engineering manipulation is possible to be conducted only on embryonic material. The aim of this study was to investigate the effectiveness of the delivery of recombinant DNA into the chicken oviduct cells in vivo and development of methodological approaches to increase the efficiency of transgenesis. In the present work a retroviral vector pLN-GFP was used, based on the Moloney murine leukemia virus (Mo-MuLV), in which a sequence of GFP (green fluorescent protein) marker gene has been cloned. Packaging cell line GP+envAM12 has been used to package retroviral vector pLN-GFP. To determine the optimal duration of administration of gene constructs a proliferative activity was analyzed in the oviduct cells at the age of 1, 2, 3, 4, 4.5 and 5 months, and at the age of 2 months within 24 and 48 hours after hormonal stimulation by 0.1 % sinestrol solution. The introduction of the retroviral vector was performed by injecting the solution of the gene construct directly into the protein part of the oviduct of hens at the age of 4 months without hormonal pre-stimulation (group I) and at the age of 2 months within 24 h after sinestrol injection (group II). Analysis of the integration and expression of recombinant DNA in chick oviduct cells was performed at the age of 6 months. As a result of histological investigations it was found that the maximum proliferative activity in the protein part of oviduct (magnum) was observed in a period between 4 and 4.5 months: the relative DNA content of the cells at this period increased by 3.4 rel. units, which was equivalent to an increase of this parameter over the entire preceding period from 1 to 4 months. The analysis of histological sections of the oviduct in 2-month-old chickens 24 hours after sinestrol injection revealed a significant change in the structure of the oviduct, typical for histological structure of the magnum portion of the oviduct at the 4 month age, and after 48 hours the oviduct histological structure corresponded to that of mature chicken. An average efficiency of the hen oviduct cell transformation assessed as a percentage of the transformed cells to the total cell number of this type in the oviduct, in group I was 17.2±3.1 % whereas in group II it was 57.3±6.3 %. Thus, the use of hormone treatment has allowed a 3.3-fold increase in the effectiveness of local transgenesis of the oviductal cells in chickens.