[Transfer of genetic constructions through the transplacental barrier into mice embryos].

Genetic modification of mammalian embryos is an important way to model various changes in human development; also, it is an instrument for studying the functions of certain genes in mammals. Using our own experience in developing modes of delivery of genetic constructions to mammals in a nonviral way, we present here data on the delivery of a eukaryotic expression vector to mice embryos through the transplacental barrier with the use of hydrodynamic intravenous injections of DNA-hybrid peptide complexes to pregnant females. The peptide has a cationic part for interaction with DNA and includes a ligand structure towards receptors of the releasing factor of luteinizing hormone (RFLH, luliberin). Advantages of the suggested method are simplicity, economy, nonimmunogenicity for females, and the ability to multiply repeat the procedure. On the basis of the method, systemic gene delivery into tissues of mammalian embryos may be developed.

[The role of ZF5 and CGGBP-20 transcription factors in expression regulation of human FMR1 gene responsible for X-fragile syndrome].

The human FMR1 gene encodes an RNA-binding protein taking part in translation regulation. The 5'-untranslated region of FMR1 gene contains a large number of tandem repeats of GCC triplets (5-50) which increasing (more then 200) is responsible for X-fragile syndrome (human congenital anomaly). As it has been shown earlier, al least two transcription factors (ZF5 and CGGBP-20) are capable of interacting specifically with GCC-repeats in regulatory regions of some genes. In this work, their roles in FMR1 gene expression regulation were studied. It was demonstrated by electrophoretic mobility shift assay that ZF5 recombinant protein specifically bound with GCC-triplet repeats (GCC9). Tissue-specific distributions of ZF5 and FMR1 proteins are very overlapped in mammalian. Inhibition of ZF5 expression in HepG2 cells (by RNA interference) leads to at least 1.5 times stimulations of FMR1 gene expression in these cells. To estimate the contribution of GCC-triplet repeats in FMR1 gene expression regulation we used two alternative variants of genetic construction: containing luciferase reporter gene under 5'-regulatory region fragment devoid of GCC-triplet repeats or including the GCC9 nucleotide sequence. HepG2 cells were co-transfected by these constructions and expressions vectors of ZF5 or (and) CGGBP-20 respectively. It was found that ZF5 downregulated the activity of 5'-regulatory region of FMR1 gene in both cases (acting probably through canonic 5'-GCGCGC3' sites). The presence of GCC-triplet repeats in the construction weakens this ZF5 effect. CGGBP-20 downregulates the activity of 5'-region of FMR1 gene in the presence of GCC-triplets only. The data obtained evidently indicate differently directed ZF5 effects on FMR1 gene expression and suggest the mechanism to explain the earlier demonstrated phenomenon about increasing of mRNA level in permutation FMR1 allele carries.

[Ap1-like cis-elements in 5'-regulatory region of human apolipoprotein A-I gene].

Several Ap1-like cis-acting elements were found within 5'-regulatory region (-2497...+173 versus transcription start point) of human apolipoprotein A-I gene (5'-apoA-I). Those elements are capable to interact with transcription factors belonging to Ap1 and CREB/ATF families. Those elements are localized outside of the hepatic enhancer (-220...-110) and the sequence responsible for apoA-I gene transcription in Caco2 cells (-595...-192). One of Ap1-like sites (5'-TGAGGTCT-3, Cre/jun2/apo) is present within 5'-apoA-I in two copies - distal (-1798 ...-1791) and proximal (+99...+106) ones. This and other Ap1-like sites - 5'-TGACTCT-3' (-1798...-1791, PF1) and 5'-TGACATCA-3' (-1171...-1163, Cre/jun1) were characterized by EMSA. It was shown by using the specific antibodies to c-Jun and ATF2 transcription factors in EMSA supershift experiments, that the DNA-protein complexes formed by Cre/jun2/apo, Cre/jun1 elements with nuclear proteins of human hepatoma HepG2 cells contain ATF2. The functional role of 5'-apoA-I regions containing Ap1-like sites was studied in cotransfection experiments of HepG2 cells (synthesize endogenous ApoA-I), human duodenum adenocarcinoma Hutu80 cells (do not synthesize endogenous ApoA-I), human neuroblastoma SK-N-SH cells (do not synthesize endogenous A-I) with expression vectors of c-jun and mekk1 genes. It was shown, that those Ap1-like sites appears to be responsible (the proximal Cre/jun2/apo is more efficient) for tissue-specific regulation of human apoA-I gene expression.

Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene.

[Delivery of "suicide" thymidine kinase gene of herpes virus in the complex with cationic peptide into human hepatoma cells in vitro]. / Dostavka "suitsidnogo" gena timidinkinazy virusa gerpesa v komplekse s kationnym peptidom v kletki gepatomy cheloveka in vitro.

The delivery of "suicide" herpes simplex virus type-1 thymidine kinase gene (tk) into tumor cells, followed by treatment with synthetic nucleotide analogues (gancyclovir, acyclovir), is a perspective approach to cancer therapy. Serious limitations in employment of the existing means of gene delivery into target cells constitute the main obstacle for cancer gene therapy development. In the present work a possibility to use a nonviral gene delivery system is shown based on the employment of lysine rich peptide K8 and amphipathic peptide JTS-1 for transferring tk gene into human hepatoma HepG2 cells. Cationic peptide K8 forms compact complexes with plasmid DNA, and JTS-1 acts as a pH-dependent endosomal releasing agent. Transfection of HepG2 cells by tk expression vector coupled with K8/JTS-1 peptides, followed by acyclovir administration (50-100 micrograms/ml) for 24 h leads to cell cycle arrest in the G1/S checkpoint of some cells, which eventually die through apoptosis. Treatment of HepG2 cells with higher acyclovir concentration (200 micrograms/ml) additionally results in a nonspecific toxic effect. The above results demonstrate the efficacy of K8/JTS-1 delivery system for the "suicide" cancer gene therapy, and may be regarded as a basis for further elaboration of "suicide" cancer approaches in vivo.