Abstract

The main goal is connected with providing, on the one hand, of active tumor-suppressor genes for prevention of eventual malignant transformations, and, on the other hand, of functionally active oncogenes for prevention of early aging and death, both in vitro and in vivo. Modulation of an adequate immune control was also necessary, and in this way any eventual unwished side effects from the genetic manipulations applied, could be escaped. Gene transfer in laboratory-cultivated mouse embryonic stem cells (mESCs) was made by use of appropriate recombinant DNA-constructs, which contained the promoter for gene, coding Elongation Factor 1-alpha (EF1-α), isolated from adeno-associated virus (AAV) (Parvoviridae); gene Dcn1, isolated from 3T3 fibroblasts of laboratory mice Balb/c, as well as gene for neomycin resistance, isolated from bacterial DNA-plasmid. Besides those indicated in the scientific literature inactivation of oncogene Dcn1 in the process of normal cell differentiation, its presence in the genome was supported and confirmed by our results from electrophorhesis of genomic DNA from normal mature epithelial cells of adult Balb/c laboratory mice. Furthermore, electrophorhetic profiles of genetic material from wild type (WT) on oncogene Dcn1 and “knock-down” (KD) on it inbred lines experimental mice differed not only on this oncogene, but also on the tumor-suppressor gene HACE1 in both categories of laboratory rodents. Similarly transfected Hela and RIN-5F malignant cells were then in vitro-co-cultivated with myeloid cell precursors, derived from populations of non-transfected laboratory-cultivated mESCs, in the presence of Doxyciclin, known from many literature data as activator of tumor-suppressor genes from STAT-family expression. Our results were also confirmed by the noticed differences in the degree of myeloid differentiation of derived precursor cells in their in vitro-co-cultivation with containing additional copies of tumor-suppressor genes malignant cells from both lines described, in comparison with the data, obtained in their laboratory co-cultivation with non-treated human cervical carcinoma Hela cells. Differences were also observed in in vitro-co-cultivation with the derived by us normal mESCs, containing additional copy of oncogene Dcn1 by the described above transfection with recombinant DNA-constructs. On the other hand, the derived normal cells with inserted additional copy of oncogene Dcn1 have indicated good safety and immunogenity. These cells have also indicated preserved normal cell characteristics, as well as eventual over-expression of the experimentally-activated oncogene Dcn1 in them.

Key words: Oncogenes, tumor-suppressor genes, myeloid cell precursors, recombinant gene constructs, cell transfection.