[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.

[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.

[Study of the transfer of foreign genes into mussel Mytilus Galloprovincialis Lam. eggs by spermatozoa]. / Issledovanie perenosa chuzherodnykh genov spermatozoidami v iaitsekletki midiii Mytilus Galloprovincialis Lam.

The possibility of transferring exogenous DNA into eggs by mussel Mytilus galloprovincialis Lam. sperms both with the use of certain methods of transfection and without them was studied. The efficacy of egg fertilization by sperms treated with foreign DNA and the development of larvae at early stages of embryogenesis were evaluated. Negative effects of the contact between mussel sperms and exogenous DNA on fertilization and subsequent development were noted. The proportion of developing larvae decreased with increasing DNA concentration and sperm exposure. Transfer of plasmids pCMVlacZ and pMTbGH into eggs was observed in group crosses. With the use of PCR, foreign DNA sequences were found in the larvae at the stage of veliger 48 h after fertilization. An intense signal was recorded after sperm electroporation in 10% DMSO.

[The transfer of foreign DNA by rabbit spermatozoa into hamster eggs]. / Perenos chuzherodnoi DNK spermatozoidami krolika v iaitsekletki khomiachka.

Capacitated rabbit sperm was incubated with pCMVlacZ plasmid and then used to fertilize hamster oocytes liberated from zona pellucida. After treatment with DNase I, these oocytes were examined for the presence of exogenous DNA by PCR. DNA of pCMVlacZ was not found in oocytes after simple incubation with male gametes. The presence of DNA was recorded in the experiments where the sperms were treated with DMSO and subjected to heat shock prior to fertilization.

[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 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.

[Amplification of the integrated bovine growth hormone transgene using the polymerase chain reaction (PCR)]. / Amplifikatsiia integrirovannogo transgena gena gormona rosta byka metodom polimeraznoi tsepnoi reaktsii (PCR).

Conditions of the PCR amplification of unique fragments of bovine growth hormone (bGH) were found that allow reliable identification of transgenic rabbits for this gene. An integrated construction containing a part of MT promoter of metallothionein gene and chromosomal bGH gene was amplified. Two of 46 newborn lambs were found to be transgenic for the bGH gene by using primers BG3 and BG4 for amplification of the gene bGH fragment and its following restriction with pVUII. We managed to whow the possibility of successful amplification of growth hormones genes of pig and sheep using primers BG3 and BG8, corresponding respectively to the beginning and the end of the bGH gene.

[The production and characteristics of transgenic mice expressing the surface protein gene of the human hepatitis B virus]. / Poluchenie i kharakteristika transgennykh myshei, ékspressiruiushchikh gen poverkhnostnogo belka virusa gepatita B cheloveka.

Transgenic mice were obtained that contained a gene of human hepatitis B surface antigen (HBsAg). The integrated HBsAg DNA sequences were inherited in the normal Mendelian fashion. 6 of 25 investigated transgenic mice expressed the HBsAg. The expression was detected in the serum and within the cytoplasm of hepatocytes. Specific tissue pathology was shown in these animals, including systemic hyperplasia of lymphoid tissue and degenerative changes in the liver and kidney parenchymatous cell elements.

[Genetic transformation of somatic cells. XI. The autonomic replication of the cloned DNA of the bovine papilloma virus in NIH/3T3 mouse fibroblasts and the changes in the growth characteristics of the transformed cells]. / Geneticheskaia transformatsiia somaticheskikh kletok. XI. Avtonomnaia replikatsiia klonirovannoi DNK virusa bych'ei papillomy v myshinykh fibroblastakh 3T3 NIH i izmenenie rostovykh kharakteristik transformirovannykh kletok.

Mouse fibroblasts NIH 3T3 were transfected with the plasmid pBPV (142-6) containing full genome of bovine papilloma virus 1, and focuses of morphological transformation were selected 2-3 weeks later. DNA molecules, containing BPV-1 sequences, were isolated from extrachromosomal fraction of transformed clones suggesting stable autonomous replication of BPV in 3T3 NIH cells. In some rescued plasmids deletions spanning E6, 7 genes of BPV were found. It is suggested that these genes are not essential for morphological transformation and autonomous replication in 3T3 NIH cells. BPV-transformed clones are able to grow in the medium containing low concentration (0.5%) of serum.

[Genetic transformation of somatic cells. X. Morphological transformation and changes in the growth characteristics of NIH 3T3 cells administered the oncogene v-myc]. / Geneticheskaia transformatsiia somaticheskikh kletok. X. Morfologicheskaia transformatsiia i izmenenie rostovykh kharakteristik kletok NIH 3T3 pri vvedenii v nikh onkogena v-myc.

Transfection of the plasmid containing the cloned gag-myc part of retrovirus MC 29 into mouse NIH 3T3 cells induces focuses of morphological transformation. Isolated morphological transformants have a decreased dependence on serum growth factors, a higher saturation density in monolayer, and an increased cloning efficiency on the glass and in agar. Induced traits are stably inherited, and may constitute the direct consequence of stable maintenance and expression of transfected oncogens.