[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.

Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 µg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 µg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

Pronuclear embryo yield in Canindé and Saanen goats for DNA microinjection.

The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 µg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.

[Mosaic expression of the lacZ reporter-gene under control of 5'-regulatory sequences of the alpha-S1-casein gene in transgenic mice].

Phenomenon of mosaic expression at cellular level is widely presented in tissues and organs of transgenic animals. The communication is concerned a study of the mosaics in transgenic mice carrying the lacZ reporter-gene under control of the bovine and goat alpha-S1-casein genes with 5'-flanked sequences of various ex-tent: pCLZ1--721bp, pCLZ2-- 2001 bp and pCLZ3 3409 bp constructs. Five transgenic founders were generated by injection of the recombinant DNA into zygotes: pCLZ 1 - N 16, pCLZ2 - N 37 and pCLZ3 N 7, N 36, and N 48. Positive for J3-galactosidase activity cells were detected in lactating mammary glands of all transgenic females, however, distribution of the positive cells was variable. We observed two types of mosaics: clonal or "lobule" type with positive cells filling the whole of the globule or stochastic type with single positive cells scattered over one or different lobules. Two types of mosaics were characteristic of all the transgenic animals, although, females carrying the pCLZ2 transgene showed "lobule" type more often than transgenic animals with the transgenes pCLZ and pCLZ3. It is suggested that the stochastic type of mosaics occurs in the cells at terminal stage of differentiation, whereas the <> type arises from positive for P-galactosidase proliferating precursors. Analysis of the inheritance of the transgenes in different lines demonstrated that the pCLZl transgene was inserted in the X-chromosome of the founder whereas the other two localized in autosomes. Localization of the pCLZl transgene in the X-chromosome did not influence the mosaicism; it was similar to that of transgenic animals carrying the transgenes in autosomes. Ectopic expression of the reporter-gene was detected in mandibular glands from the offsprings of the founders N 16 and N 37 only, as well as in atrezed follicles in N 37. The weak ectopic expression saggests that the 5 S-flanked regulatory sequences used in the constructs are able to provide perfect tissue-specific expression of the reporter-gene.

[The effect of regulatory sequences of alphaS1-casein gene on the expression of the lacZ-gene in loach Misgurnus fossilis L. transgenic embryos].

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine alphaS1-casein gene tissue-specific promoter-enhancer region or highly homologous goat alphaS1-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1-3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the alphaS1-casein gene of goat was higher than with the promoter-enhancer region of the bovine alphaS1-casein gene. Thus, regulatory sequences of the bovine or goat alphaS1-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.

[Analysis of transfer of microinjected zygotes in production of transgenic mice].

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients' pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.

[Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice].

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5' and 3' flanking promoter sequences of bovine alpha-S1-casein gene. Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3' flanking region of bovine gene @CSN1S1, but differed in size of the 5' flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 microg/ml to over 1 mg/ml, in mice with construct pGCml and was low (up to 60 microg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCml and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF.

[Analysis of the formation and autonomous replication of an extrachromosomal mouse transgene]. / Analis protsessov formirovaniia i avtonomnoi replikatsii ékstrakhromosomnogo transgena myshei.

A stable autonomously replicating shuttle transgene, pr8a was, previously isolated from Bombix mori and characterized. Autonomous replication of pr8a and its derivatives was observed in yeast cells, B. mori embryos, and transgenic mice. To continue the previous studies, transgenic mice of several generations were examined. DNA analysis revealed a course of pr8a rearrangements resulting in extrachromosomal transgene p8-2 of F2 transgenic mice. Consecutive directional elimination of vector DNA fragments was characteristic of rearrangements affecting autonomous transgenes in mouse cells. Analysis of the data obtained with transgenic mice and other organisms made it possible to identify the pr8a fragment that acts as minimal ARS in transformed yeast cells and transgenic mice. Another pr8a region was assumed to be a component of a pr8a ARS module involved in regulating autonomous replication. Inserted in two different integrative vectors, minimal ARS ensured their autonomous state in transformed yeast cells and transgenic mice. Transgenes pr8a and p8-2, along with some other well-known constructs, were considered as prototype autonomously replicating vectors suitable for studying the mechanism of autonomous replication and solving some problems of gene therapy.

DNA polymerase iota-like activity in crude cell extracts of different mouse organs.

The recently discovered DNA polymerase iota differs greatly from the numerous eukaryotic and prokaryotic DNA polymerases known previously in its ability to catalyze error-prone DNA synthesis. Using homogeneous preparations of the enzyme, it was shown previously that DNA polymerase iota incorporated preferentially dGMP opposite the thymidine of the template in the growing DNA chain. To elucidate the role of this enzyme in the mammals, its activity was assayed in crude cell extracts of different mouse organs. It is shown that the extracts of the brain and testis cells exhibit the highest activity of DNA polymerase iota, which is not in agreement with the results of other authors. The data suggest that the tissue specific expression of DNA polymerase iota is regulated to a significant degree at the posttranscriptional and posttranslational levels.

[Viral promoter-enhancer element inhibits replication of autonomously replicating plasmid in mammalian cells]. / Virusnyi promotorno-énkhansernyi élement ingibiruet replikatsiiu avtonomno replitsiruiushchikhsia plazmid v kletkakh mlekopitaiushchikh.

To study the autonomous replication of constructs combining replication and transcription elements, hybrid plasmids were designed to contain both higher eukaryotic ori/ARS and the cytomegalovirus (CMV) promoter-enhancer. In pCI.ARS-neo, ARS was from autonomously replicating stable transgene pr8a of silkworm Bombyx mori. In pCI.ori-neo, chromosomal ori was from the human c-myc locus. Analysis of the maintenance of pCI.ARS-neo and pCI.ori-neo in transgenic mice and in cultured immortalized human T cells revealed the interplay of transcription and replication, as the CMV element suppressed autonomous replication of both plasmids. This might reflect the conflict between transcription and replication of the genome in higher eukaryotic cells.

[Loach spermatozoa transfer foreign DNA, which expression is discovered in the early development stages]. / Spermatozoidy v'iuna perenosiat chuzherodnuiu DNK, ékspressiia kotoroi obnaruzhivaetsia na stadiiakh rannego razvitiia.

The transfer of plasmid pcDNA3-lacZ by electrotrasfected sperm cells into loach (Misgurnus fossilis L.) ova has been studied. The lacZ gene has been found to express in 3- to 5-day-old prelarvae.

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil.

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.

[Targeting the gene for platelet-derived growth factor A-chain (PDGF-A) and preparation of chimeric mice with a "knockout" genome]. / Targeting gena A-tsepi trombotsitarnogo faktora rosta (PDGF-A) i poluchenie khimernoi myshi s "nokautirovannym" genom.

A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.

[Effects of the nef and tat genes of the human immunodeficiency virus type I on rodent cells in vivo and in vitro]. / Vliianie genov nef i tat virusa immunodefitsita cheloveka tipa 1 na kletki gryzunov in vivo i in vitro.

The tat and nef regulatory genes of the human immunodeficiency virus type I (HIV-1) under the control of eukaryotic promoters were transferred in vivo into mice and in vitro into rat cell cultures. The development was disturbed and adenocarcinomas of the lacrimal glands and pancreas appeared in transgenic mice carrying the HIV-1 tat gene. Transfection with the tat gene altered morphology and increased proliferative activity of Rat-2 pseudonormal cells. The tat gene also induced the formation of neoplastic foci in a primary rat embryo fibroblast culture. The results obtained showed that the HIV-1 tat gene can act as an oncogene and activate the proliferation of cultured cells. Cell proportions in peripheral blood and bone marrow were altered and mitogen-induced lymphocyte proliferation was decreased in transgenic mice carrying the HIV-1 nef gene. This gene also significantly suppressed proliferation but had no effect on morphology of Rat-2 cells. Thus, the HIV-1 nef gene appeared to suppress proliferation of various animal cells.

[Expression of the CMV-lacZ- and RSV-lacZ-genes in transgenic fish and mouse embryos]. / Ekspressiia CMV-lacZ- i RSV-lacZ-genov v transgennykh émbryionakh ryb i myshei.

The bacterial gene for beta-galactosidase under the control of LTR from either human cytomegalovirus (CMV-lacZ) or the Rous sarcoma virus (RSV-lacZ) was injected into fertilized eggs of Misgurnus fossilis L. loaches and F1 hybrid mice (CBA x C57Bl). Expression of the CMV-lacZ was observed in almost 100% of the loach embryos and larvae for two months following the first day of embryonic development. In some cases, expression points were located only on either the right or the left side of a fish. The spectrum of tissues expressing CMV-lacZ was decreased during embryonic development: CMV-lacZ was expressed only in fin and body muscles of 6- to 8-week-old loaches. In mice, the RSV-lacZ gene was expressed in ectoderm- and mesoderm-derived tissues of a 13-day-old embryo, and the CMV-lacZ gene was expressed in tissues derived from various blastophylli of a 14-day-old embryo. Distribution of transgene expression is discussed with regard to authors' and published data.

[Analysis of expression of foreign genetic material under the control of heterogeneous promoters in the process of early development of the loach (Misgurnus fossilis L.)]. / Analiz ékspressii chuzherodnogo geneticheskogo materiala pod kontrolem geterogennykh promotorov v protsesse rannego razvitiia v'iuna (Misgurnus fossilis L.).

Promoters normally active in the nuclei of evolutionarily distant from fishes taxa-bacteria, insects, and Protozoa-are shown to be able to regulate heterologous genes during early development of the loach. It is shown that particular DNA sequences can drastically influence the function of eukaryotic promoters, enhancing the expression level.

[Analysis of expression of the RSV-lacZ-gene in transgenic embryos of the loach Misgurnus fossilis L. during different variations of injection]. / Analiz ékspressii RSV-lacZ-gena v transgennykh émbrionakh v'iuna Misgurnus fossilis L. pri razlichnykh variatnakh in''ektsii.

Several series of microinjection of the RSV-lacZ gene (the Escherichia coli beta-galactosidase gene under the control of the long terminal repeat of the Raus sarcoma virus) into fertilized mud loach eggs were carried out. The expression of the transgene in 3-5 day-old fry was shown to depend neither upon the stage of fry development at which the RSV-lacZ gene was introduced (early blastodisc, late blastodisc, 2-blastomere embryo) nor upon the region of transgene injection (blastodisc cytoplasm or egg yolk). Additionally, when injected into yolk, a smaller number of transgene expression points were observed and their distribution was confined to the surface of the yolk sac. In some experimental series, transgene expression was observed in 100% of embryos. The presence of exogenous genetic material in one of the experimental series was proved for 100% of 5- to 6-week old juveniles, when injected into cytoplasm, and for 67% of juveniles, when injected into yolk. This work provides evidence of the possibility of 100% transfer and expression of exogenous DNA when transgenic loachs are generated by injection into embryos and fry at different stages of their development.