[Effect of Ultrasmall Gold Nanoparticles on the Murine Native Sperm Chromatin].

The effects of ultrasmall (2-3 nm) gold nanoparticles on native epididymal sperm chromatin of CBAxC57BL/6 hybrid mice and 129/IMG mice with a mutation in the DNA-polymerase iota gene were studied. It is shown that for both mouse strains after sperm incubation in a solution containing Au nanoparticles, at 23, 37 and 60 degrees C for 30 min followed by 1 hour treatment in dithiothreitol solution, a decrease in the number of nuclei with fully decondensed chromatin was observed compared with the control. Though, the manifestation of this effect in the population of 129/IMG mice mature sperm, was weaker. Also we have demonstrated that sperm of both strains that were incubated in a sol of Au nanoparticles at 60 degrees C behave differently under the action of dithiothreitol. A considerable part (-80%) of sperm of CBAxC57BL/6 hybrid mice treated with Au nanoparticles showed high resistance to the action of dithiothreitol, whereas in the case of 129/IMG mice only -30% did, and a partial or complete chromatin decondensation takes place in the remaining sperm. In general, using the method of nuclear chromatin decondensation in vitro for the native sperm, the patterns that we have identified in earlier studies on previously demembranized sperm are confirmed.

[Bovine sperm chromatin is not protected from the effects ultrasmall gold nanoparticles].

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of 3.0 nm and a concentration of 1 x 10(15) particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed ("intact"), partially decondensed, and completely decondensed nuclei correlated as 40 : 32 : 28% and 0 : 36 : 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.

[Effect of gold nanoparticles on mouse spermatogenesis].

The response of the mouse male germ cells exposed to gold nanoparticles (approximately 2.5 nm) was studied. Our investigation demonstrates that treatment with Au nanoparticles for four days does not impair the architecture of the spermatogenic epithelium. Cytogenetic evaluation using micronucleus assay showed that gold nanoparticles can affect the chromosomes of early primary spermatocytes. However, gold nanoparticles did not induce chromosome abnormalities in spermatogonial stem cells. Further, the cauda epididymal sperm was isolated on the 14th day after treatment and was incubated in SDS solution (Na sodium dodecyl) and then in a solution containing DTT (dithiothreitol) to induce nuclear chromatin decondensation. Observations showed that after four days of treatment of spermiogenic (postmeiotic) cells with gold nanoparticles the decondensation process had no differences from the control. On the contrary, in the experiment with the same cells and period of fixation but with a single exposure to gold nanoparticles, the number of mature gametes with totally decondensed nuclei reached 100% as opposed to 44% in the controls.

[Advanced approaches to studying the population diversity of marine fishes: new opportunities for fisheries control and management].

Recent conceptual and technological advances now enable fisheries geneticists to detect and monitor the dynamics and distribution of marine fish populations more effectively than ever before. Information on the extent of genetically-based divergence among populations, so-called "population diversity", is crucial in the quest to manage exploited living resources sustainably since it endows evolutionary potential in the face of environmental change. The generally limited dialogue between scientists, fisheries managers and policy makers, however, continues to constrain integration of population genetic data into tangible policy applications. Largely drawing on the approach and outputs from a European research project, FishPopTrace, we provide an example how the uncovering of marine fish population diversity enables players from genetics, forensics, management and the policy realm to generate a framework tackling key policy-led questions relating to illegal fishing and traceability. We focus on the use of single-nucleotide polymorphisms (SNPs) in European populations of cod, herring, hake and common sole, and explore how forensics together with a range of analytical approaches, and combined with improved communication of research results to stakeholders, can be used to secure sufficiently robust, tractable and targeted data for effective engagement between science and policy. The essentially binary nature of SNPs, together with generally elevated signals of population discrimination by SNPs under selection, allowed assignment of fish to populations from more areas and with higher certainty than previously possible, reaching standards suitable for use in a court of law. We argue that the use of such tools in enforcement and deterrence, together with the greater integration of population genetic principles and methods into fisheries management, provide tractable elements in the arsenal of tools to achieve sustainable exploitation and conservation of depleted marine fish stocks.

[Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain].

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.

[Analysis of spermatogenesis in senescence-accelerated mice].

A comparative analysis of age-related dynamics of spermatogenesis has been performed in mutant mouse lines predisposed or resistant to accelerated senescence (SAMP1 and SAMR1 respectively). The results show that quantitative and morphohistological trends in the development of sperm cells and Sertoli cells in both lines are similar in both lines. Their comparison with data obtained in our previous studies (Zakhidov et al., 2001; Gordeeva et al., 2001) shows that sharp quantitative and qualitative changes in the structure of the spermatogenic system have occurred in senescence-accelerated mice of new generations, which confirms the fact of dynamic instability of the germinal lineage. The role of stem spermatogonial cells in restoration of spermatogenesis in animals reaching the critical age is discussed.

[Hereditary muscular dystrophy: bioengineering approaches to muscle fiber repair].

Restoration of disturbed functions of the organs and tissues is the main task of contemporary genetic and cellular biotechnology, including genetic and cellular therapy. Duchenne dystrophy, one of the most widespread human genetic diseases, is at the same time the most extensively studied from the viewpoint of both genetic and histological changes leading to muscle fiber degeneration. Although many studies carried out on models, recognized analogous to Duchenne dystrophy, gave hopeful results, clinical tests with the use of developed methods gave no expected success and the rate of mortality from this disease amounts to 100%. Based on the world experience and analysis of the authors' data, possible influence of the intensity of regeneration on success of genetic and cellular therapy has been considered.

[The BCL-xL and ACR-1 genes promote differentiation and reduce apoptosis in muscle fibers of mdx mice]. / Geny BCL-xL i ACR-1 sposobstvuiut differentsirovke i umen'shaiut gibel' myshechnykh volokon myshei mdx.

The effects of the human BCL-xL and ACR-1 genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. Introduction of the dystrophin minigene (20 micrograms of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 +/- 1.4% and 4.5 +/- 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 micrograms of each plasmid per shot), the death of CSMF decreased to 3.7 +/- 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 +/- 2.2%. Contransfection with the dystrophin minigene and the BCL-xL gene at 20 micrograms of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 +/- 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 micrograms of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 +/- 0.5% and significantly reduced the proportion of dying CSMF to 0.9 +/- 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human-dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xL or ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.

[Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice]. / Gen hACR-1 podavliaet apoptoz poperechnopolosatykh myshechnykh volokon m.quadriceps femoris myshei mdx pri ballisticheskoi transfektsii.

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.

The quantitative characteristics of efficiency of ballistic transfection of chimeric antibody genes.

A quantitative approach was applied to the study of in vivo expression of foreign genes introduced into mice by ballistic transfection. Because in some cases one must take into account both the level of synthesized protein and that of antibodies to it, we derived the equation which allows to calculate the exact quantity of both proteins. This formula was applied to in vivo expression of a chimeric (human/mice) immunoglobulin E gene. The immunochemical analysis using this equation showed that the Ig concentration succeeded 4, 6, 12 IU/ml and undetectable level, respectively, upon transfection in mouse liver, spleen, foot pad and ear cartilage.

[Activity of exogenous genetic designs, introduced into cells by a ballistic transfection method, in murine ontogenesis]. / Aktivnost' ékzogennykh geneticheskikh konstruktsii, vvedennykh v kletki metodom ballisticheskoi transfektsii, v ontogeneze myshi.

We used the method of particle bombardment (ballistic transfection) to introduce beta-galactosidase and human dystrophin genes into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells, the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective, convenient, and practical methods of skeletal muscles transfection.

[Differentiation of muscle fibers in mdx mice after ballistic transfection of cDNA of the human dystrophin gene]. / Differentsirovka myshechnykh volokon myshei MDX posle ballisticheskoi transfektsii kDNK gena distrofina cheloveka.

Changes in morphological dimensions of MDX mouse myofibres in M. rectus femoris were recorded after ballistic transfection (BT) with pHSADys and pVMMDys plasmids containing cDNA of the full-length human dystrophin gene. The dystrophin expression was observed by an immunomorphological procedure with P6 antibody and PAP method. Dystrophin positive (dyst+) myofibres were divided into two types, with a typical dystrophin expression under sarcoplasma membrane and an atypical expression through the whole sarcoplasm, respectively. The share of atypical dyst+ myofibres was seen to rise during the experiment from 27%, at 2-3 weeks after BT, up 84% by 2 months after BT. The atypical dyst+ myofibres usually underwent destruction. At the same time, the share of entire dyst+ myofibres decreased from 17 to 2-5% by 2 months. Morphological dimensions of the myofibres (square in mkm2, perimeter, smallest and largest diameters) were calculated with the help of computer analyser. The middle square of both types of dyst+ myofibres was larger than that of dyst- myofibres, both in BT target M. rectus femoris and in the same contralateral muscle, but never exceeded the value of middle square of C57B1 mouse myofibres in the same muscle. The form of dyst+ myofibres was not modified by the dystrophin expression. The nuclei of dyst+ myofibres remained in the central region of sarcoplasm. A conclusion is made that BT of human dystrophin gene inside MDX mouse myofibres allows dystrophin gene expression and enlargement of the dyst+ myofibres. Dystrophin expression is not able to induce a complete and stable differentiation of striated muscle of adult MDX mice.

[Expression of the human dystrophin gene in mdx mouse skeletal muscles after ballistic transfection]. / Ekspressiia gena distrofina cheloveka v skeletnykh myshtsakh myshei mdx posle ballisticheskoi transfektsii.

"Gene-gun" ballistic transfection (BT) was used to deliver genetic constructs pMLVDy and pHSADy containing full-length cDNA of the dystrophin gene to musculus quadriceps remoris and musculus gluteus of mdx mice, which represent a natural model of Duchenne muscular dystrophy. Clusters of dystrophin-positive muscular fibers (DPMF) were immunocytochemically detected in sites exposed to BT. The average number of DPMF was 2% by the 17th day and 3% by the 60th day after BT with pMLVDy, whereas the number of revertant DPMF was 0.2% in control mice (without BT). When pHSADy was used, the average number of DPMF was 3% 20 days after BT. In this case, dystrophin was uniformly spread though the myoplasm in 3% of cells and produced a slight signal in separate regions under the sarcolemma in 10% of muscle fibers. The number of revertant DPMF increased to 0.6% after BT with naked particles and to 2.8% after BT with the marker lacZ gene, in both bombarded and contralateral legs. The number of DPMF in the corresponding muscles of the contralateral leg significantly increased and reached 2.8% by the 60th day after BT with pMLVDy and 6.7% by the 20th day after BT with pHSADy. Human dystrophin gene cDNA was detected in all skeletal muscles, heart, intestine, tongue, and brain by polymerase chain reaction (PCR) three weeks after BT. Immunoblot analysis showed that normal 427-kDa human dystrophin was synthesized in muscles of mdx mice. The results suggest applicability of BT for delivery of dystrophin constructs into muscles.

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

[Metallonucleoliposome complexes as a vehicle for gene delivery to mouse skeletal muscles in vivo]. / Metallonukleoliposomnye kompleksy kak sredstvo dostavki genov v skeletnye myshtsy myshi in vivo.

A simple new method for preparing plasmid DNA and preformed zwitterionic liposome complexes is proposed. The ability of these metallonucleoliposome complexes to serve as a vehicle for gene delivery to mammalian cells in vivo was studied. A high level of expression of the reporter gene introduced was observed in mouse skeletal muscles in vivo.

[The ballistic transfection of mammalian cells in vivo]. / Ballisticheskaia transfektsiia kletok mlekopitaiushchikh in vivo.

The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Having penetrated in the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized and this allowed to study mammalian cells in situ. The studies performed allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ and in developing mouse embryos at the stages of two-cell embryo, morula and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was found within two-three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail and the published data on transfection and genetic transformation of mammalian cells are discussed.

Transfer of foreign DNA into the cells of developing mouse embryos by microprojectile bombardment.

Mouse cells of developing embryos at the 2-4 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot-blot hybridization and PCR with post-hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3-neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418-resistant clones.

[Use of high velocity mechanical injection for the transfer of foreign DNA into early mouse embryos]. / Ispol'zovanie vysokoskorostnoi mekhanicheskoi in"ektsii dlia perenosa chuzherodnoi DNK v rannie émbriony myshei.

The possibility of high velocity mechanical transfer of foreign DNA into inner cell mass of mouse blastocyst was shown. Penetration of tungsten microparticles into early embryo cell nuclei and their localization on mitotic chromosomes was demonstrated. About 70% of developing embryos survived the bombardment. Total DNA of the mice born from bombarded embryos was analyzed by blot-hybridization and PCR with Southern hybridization. In three cases, the presence of the transferred plasmid DNA (pSV3-neo) was revealed.