Generation of LEPR Knockout Rabbits with CRISPR/CAS9 System.

The LEPR gene encodes a leptin hormone receptor, and its mutations are associated with morbid obesity, dysregulation of lipid metabolism, and fertility defects in humans. Spontaneous Lepr mutations have been described in rodents, and Lepr knockout animals have been generated, in particular, using the CRISPR/Cas9 system. Lipid metabolism in rodents significantly differs from that in humans or rabbits, and rabbits are therefore considered as the most relevant model of morbid obesity and lipid metabolism dysregulation in humans. LEPR knockout rabbits have not been reported so far. In this work a LEPR knockout rabbit was generated by introducing a deletion of the region around LEPR exon 10 using the CRISPR/Cas9 system. The body weight of the knockout rabbit was significantly higher than the average body weight of the wild type rabbits. CRISPR/Cas9-mediated generation of LEPR knockout rabbits will allow the development of a model of morbid obesity and endocrine defects due to leptin receptor mutations in humans.

Melanophilin Polymorphism in Ferrets of Different Color.

In mammals, the main contribution to the variability of pigmentation is made by two groups of genes directly related to the metabolic pathways of pigment synthesis and controlling the transport of melanosomes in melanocytes to keratinocytes. In order to identify the genetic basis of pigmentation variants, the nucleotide sequences of the melanophilin gene were compared in two groups of ferrets-silver-colored and wild-type animals-using sequencing of 16 exons. In carriers of silver color, a single nucleotide deletion was detected in the 9th exon, leading to a shift in the reading frame and the formation of a stop codon downstream. The protein encoded by the mutant allele is almost completely devoid of the C terminal domain of the protein responsible for the contact of melanosomes with actin during their moving to the periphery of melanocytes, but it retains the leading domain involved in the formation of melanosomes. The combination of the preservation of the N domain and the defect of the C domain of the mutant protein for the first time makes it possible to explain the incomplete dominance of the wild-type protein in heterozygotes.

Sources of contradictions in the evaluation of population genetic consequences after the chernobyl disaster.

The review covers the analysis of our own and published data pertaining to population and genetic consequences in various mammalian species under conditions of high levels of ionizing radiation as a result of the Chernobyl accident. The findings indicate that these conditions have promoted the reproduction of heterozygotes in polyloci spectra of molecular genetic markers and animals with a relatively increased stability of the chromosomal apparatus. The prospects of using the reproductive "success" of the carriers of these characteristics as an integral indicator of the selective influence of environmental stress factors are discussed.

Karyotype alterations in human lung adenocarcinoma cells after long-term action of interferon-alpha.

AIM: To estimate the effect of long-term IFN treatment of human non-small-cell lung cancer cell line A-549 on their karyotype characteristics and on the clonal structure of cell population. METHODS: Cytogenetic research was performed by standard methods using routine and differential staining. Cytogenetic characteristics were estimated per 1000 cells (ppm, (per thousand)). RESULTS: Cytogenetic analysis of IFN-modified A-549 human lung cancer cells had demonstrated far-going changes in their population structure. It was shown that long term cultivation with IFN altered the chromosome modal class of A-549 cells, induced the domination of hromosomes with certain molecular markers: the number of metaphases with der (6) t (6; 1) chromosomal rearrangement increased significantly (from 6% to 80%, p CONCLUSION: Long-term IFN effect results in alterations of cytogenetic properties of A-549 human lung cancer cells.

Spontaneous premature condensation of chromosomes in normal and transformed mammal cells.

AIM: To study the relation between premature chromosome condensation and the ability of the cells to undergo malignant transformation. METHODS: Standard cytogenetic analysis of bone marrow cells and cultured normal and tumor cells has been used. RESULTS: Comparative analysis of the frequency of occurrence of the cells with premature chromosome condensation (PCC) (cell "arrest" at G2/M phase) in relation to dividing cells in the cultures of human immortalized cells of hematopoietic origin, human lung carcinoma A-549 cells, and in populations of bone marrow cells of BALB/c and C57BL/6 mice differing in predisposition for myeloma development has been performed. It has been revealed that in populations of bone marrow cells of C57BL/6 mice the relation of cells with PCC to dividing ones is 2-3-fold lower than in other studied cell populations. Immortalized and malignantly transformed human cell lines were characterized by high frequency of occurrence of cells with PCC. In the cells of A-549R subline characterized by suppressed malignant phenotype this index was lower than in parental A-549 cells. CONCLUSION: The obtained data point on possible relation between disturbed passing of "check point" by cells upon transition from G2 phase of cell cycle to mitosis and increased genetic heterogeneity of new cell generation associated with ability of cells to immortalization and malignant transformation.

[Chronic low doze ionizing irradiation and polyfactors of adaptation].

The analysis of cytogenetic anomaly dynamics in conditions of chronic low doze effect of ionizing irradiation in generations of genetically homogeneous laboratory line of mice CC57W/Mv and genetically heterogeneous populations of vole's species, trapped in alienation zone of Chernobyl's NPP in places with different levels of radio nuclide contamination in 1994-2001 was carried out. The data about the physiological adaptation to ionizing irradiation effects under age of linear mice and non-linearity of frequency changes of separate cytogenetic anomalies in generations as laboratory line of mice, and genetically heterogeneous populations of vole's species were obtained. Was supposed, that the number of non-linear effects of the ionizing irradiation in low doze range could be stipulated by comparability between the intensity of it's damage effects and the activation by it the polyfactor systems of anti oxidant ones, reparation events, and also elimination of defective cells.

[The possible ways of the diploidization of the polyploid germinative stem cells in the mouse BALB/c line].

Quantitative and qualitative chromosome rearrangements in the cell line G1 established from a genital ridge of the 12,5 dpc BALB/c mouse embryo were analysed. Cytogenetic analysis was performed on the 75th passage of in vitro cultivation. It has been shown that by this passage the cell population was heterogenous. It is suggested that such heterogeneity may be caused by realization of two simultaneous processes namely the cell polyploidization and their secondary diploidization. These processes were accompanied by some chromosome destructions, and the creation of small new acrocentric chromosomes and large aberrant chromosomes as well as Robertsonian translocations. The present study demonstrates in vitro karyotype evolution of the mouse cell line G1 including the increased instability of the chromosome apparatus.

[Spontaneous mutagenesis in bone marrow cells in the CBA and CBA/H-T6 mouse strains].

Comparative analysis of cytogenetic characteristics in bone marrow cells of the mouse lines CBA and CBA/H-T6 has been carried out. It was shown that translocation T6 effects the apparatus of cell division and can cause additional cytogenetic abnormalities.

["Chain" karyotypic evolution of embryonic stem cell line R1 in vitro].

Cytogenetic anomaly frequencies were analysed in three sublines of ES R1 line in its five clonal sublines, obtained from two cell colonies after transformation of ES R1 cells by plasmid with gene lif. Cell transformation did not increase cytogenic anomalies, however, the initial sublines of ES R1 line, as well as its transformed clonal descendants bore a redundant quantity of the chromosome 8 material within the structure of various Robertsonian translocations even in cells with diploid chromosome quantity (2n = 40). In the initial sublines ES R1 and its clonal descendants a common Rb (8; 15) was revealed. It was supposed that selection for the increase in ES cell sensitivity to cytokines (in particular, LIF) under cultural conditions was accompanied by an increase in chromosomal copies, carrying genes of mapk andjak/stat, through which downstream effectors of cytokine signals for preservation of cell pluripotention and propagation are realized. Genes of chromatid separation and chromosome segregation control (for example, separase gene Esp1 in chromosome 15) may be passively involved in this process, thus promoting acceleration of karyotype evolution in ES cells.

[Species specificity of karyotype instability under conditions of radionuclide contamination (the Chernobyl Nuclear Power Plant) in the voles, Micrototus oeconomus, Micrototus arvalis, and Clethrionomys glareolus]. / Vidospetsifichnost' destabilizatsii karotipa v usloviiakh radionuklidnogo zagriazneniia (ChAES) u polevok Microtus oeconomus, Micrototus arvalis, Clethrionomys glareolus.

The comparative analysis of frequencies of different type cytogenetic anomalies in voles Microtus oeconomus, Microtus arvalis, Clethrionomys glareolus trapped in territories with different levels of radionuclide contamination inside the 30-km zone of estrangement around the Chernobyl NPP was carried out. Animals with constitutive chromosome abnormalities were not revealed. The frequency of lympocytes with micronuclei was the most universal and sensitive character to chronic low-dose radiation in investigated species. The species-specific relation was observed between level of radionuclide contamination in trapping zone and increase in frequencies of cytogenetic abnormalities: aneuploidy in Microtus arvalis and metaphase plates with asynchronous centromere fission in Microtus oeconomus. Common vole appears to be the most sensitive species of investigated ones to chronic low-dose ionizing irradiation.

[The interrelations between karyotypic stability and interchromosomal associations in mammalian cells]. / Izuchenie vzaimosviazei mezhdu kariotipicheskoi stabil'nosti i mezhkhromosomnymi assotsiatsiiami v kletkakh mlekopitaiushchikh.

The interchromosome associations between heterologous chromosomes (for the type of Robertsonian translocations); frequencies of the association between individual chromosomes (identified by G-bands) and mouse line pecularities, the directions of cytodifferentiation, the stages of cell neoplastic transformation in cell populations of various origin have been estimated. The presence of interrelations between individual chromosome associations and genetic and cytological cell characteristics with the use of different mathematical methods of analysis has been revealed.

[The heterogeneity of the cytogenetic variability in the bone marrow cells of laboratory and wild rodents in the exclusion zone of the Chernobyl Atomic Electric Power Station]. / Geterogennost' tsitogeneticheskoi izmenchivosti v kletkakh kostnogo mozga laboratornykh i dikikh gryzunov v usloviiakh zony otchuzhdeniia Chernobyl'skoi AES.

Analysis of the characters of cytogenetic variabilities and correlative interrelations between them revealed the group specificities of karyotype instability in bone marrow cells of some lines of laboratory and of wild mice under the conditions of increased radionuclide pollution in the Chernobyl zone. The complex correlative interrelations between the frequencies of cytogenetic anomalies and the character of cell division (the number of metaphases, binucleated leukocytes per 1000 cells) were observed. The obtained data indicate the stimulatory influence of chronic low doses of ionizing radiation on the manifestation of spontaneous mutagenesis, which has own genotype specific characteristics.

[The variability of different cytogenetic characteristics in groups of Black Pied cattle]. / Izmenchivost' razlichnykh tsitogeneticheskikh kharakteristik u grupp cherno-pestrogo skota.

The comparative analysis of frequency of occurrence of different indications of cytogenetic variabilities in blood cells of cattle groups (relative healthy, infected by bovine leukosis virus and exposed to ionizing radiation in the Chernobyl zone) was carried out. Both genotoxic influences induced increases in frequencies of cytogenetic defects, but only ionizing radiation induced the increase of chromosome aberration frequency. The heterogeneity of all cattle groups for investigated cytogenetic characters was revealed.

[The independent variability of different karyotypic characteristics during the spontaneous neoplastic evolution of mouse embryo fibroblasts]. / Nezavisimaia izmenchivost' razlichnykh kariotipicheskikh kharakteristik v protsesse spontannoi neoplasticheskoi évoliutsii émbrional'nykh fibroblastov myshei.

The dynamics of some karyotype characteristics throughout the spontaneous neoplastic evolution of embryonal fibroblasts of different mouse strains was studied using a differential staining of chromosomes. The independent variability of chromosome modal numbers, the number of polyploid cells in the cell line, the number of chromosomal rearrangements within one cell, the activity of formation of different types of chromosomal rearrangements and the activity of interactions of the homologous chromosomes in 15 studied cell lines were revealed. Possible levels of variability regulations of different karyotype characteristics are discussed.

[Hematologic chimerism in cattle twins]. / Gematologicheskii khimerizm u bliznetsov krupnogo skota.

A cytogenetic examination of a cow and its offsprings, obtained by pair transplantations of the embryos was carried out. The fragile X and the heteromorphism of the chromosomes XX of this cow and some of its offsprings were revealed. The variability of the hematological chimerism of the pair transplanted embryos was observed. The immigrated cells were, as a rule, distinguished by the comparatively low frequencies of the metaphases with the nearly located chromosomes XX. The heterogeneity of the stem blood cells of the embryos for some traits, and for the ability to migrate in the blood of the twin is under discussion.

[The spontaneous aneuploidy of the embryonic fibroblasts in C3H/He and CBA/Ca mouse strains occurring during their neoplastic evolution]. / Spontannaia aneuploidiia émbrional'nykh fibroblastov myshei linii C3H/He i CBA/Ca, voznikaiushchaia v protsesse ikh neoplasticheskoi évoliutsii.

Karyological analysis of 6 cell lines with distinct tumorigenic properties of mouse strains C3H/He and CBA/Ca has been carried out using differential chromosome staining. All the cell lines are characterized by a decreased number of copies of normal chromosome 7, the increased number of normal copies of chromosome 10 being specific of the cell lines with intermediate tumorigenicity. Cell lines with maximum tumorigenicity differed from all other lines by the increased number of copies of chromosome 5 and by the decreased number of copies of chromosome 6. A wide independent variability was observed in the number of chromosomes and of several types of abnormal chromosomes throughout the neoplastic evolution of cells, to begin from the early immortal passages. But the proportion of normal chromosomes per cell in the studied lines revealed relatively stable values. The potential phenotypical heterogenicity of the lines with maximum tumorigenicity, expressed in their clonal progeny, was associated with the instability in the number of chromosome 15 copies in cells of these lines. It is concluded that multiple genetic events are required in the spontaneous neoplastic evolution of fibroblasts, and only specific traits of the karyotypic instability, associated with the variability of the number of copies of specific chromosomes, may constitute the genetic basis for the above process.

[The karyotypic variability of mouse embryonic fibroblasts of 2 genotypes during spontaneous neoplastic transformation]. / Kariotipicheskaia izmenchivost' émbrional'nykh fibroblastov myshei dvukh genotipov v protsesse spontannoi neoplasticheskoi transformatsii.

Karyological analysis of 6 lines with distinct tumorigenic properties of mouse strains C3H/He and C57BL/6 has been carried out using a differential staining of chromosomes. The number of normal copies of chromosomes varied in all the investigated cell lines. The more and the less stable chromosomes different from line to line. All the cell lines were characterized by decreased numbers of copies of normal chromosome 7; a decreased number of normal copies of chromosome 2 and 16 was detected in the course of the cell spontaneous neoplastic evolution. The decreased number of normal copies of chromosomes 8, 12 and X, and the increased number of normal copies of chromosome 10 were specific of the cell lines with intermediate tumorigenicity. The maximum tumorigenic cell lines differed from all other lines by increased numbers of copies of chromosomes 4 and 5, and by a decrease in copy number of chromosome 6. The data obtained are discussed in terms of the search of the regularity of karyotypic changes in the course of the cell neoplastic evolution.

[Approaches to the analysis of reciprocal chromosome distribution in the bone marrow cells of mice]. / Podkhody k analizu vzaimnogo raspolozheniia khromosom v kletkakh kostnogo mozga myshei.

A study was made of G-banding chromosome preparations made of bone marrow cells of BALB/c mouse females, injected intraperitoneally with a single dose of the antibiotic adriamycin 23 hours before sacrifice. Frequencies of different chromosome fusions in pericentromeric regions were analysed. All chromosomes were found to be involved in the interchromosomal associations, different chromosomes participating in such fusions with different frequency. No associations of homologous chromosomes were observed. For each chromosome a limited number of preferential partners for fusion was revealed, the number of such partners for different chromosomes varying. Almost for a half of chromosomes, chromosome 15 was one of the most preferred partners. Possible significance of the data obtained is discussed for the analysis of principles of the spatial chromosome arrangement in murine bone marrow cells.

[Karyotypic characteristics of a number of clonal lines of an interspecific mouse-mink hybridoma]. / Kariotipicheskie osobennosti riada klonal'nykh linii mezhvidovoi gibridomy mysh'--norka.

Using G-banding method, a study was made of the karyotypes of mouse myeloma cell line P3-X63-Ag8.653 and some cell hybrid lines originated from a fusion of mink immunized spleen cells and this myeloma. Normal chromosomes 6, 12 and X were not detected in either examined cell of the parental myeloma. The mink chromosomes are preferentially lost from cells of the hybrid lines. These lines varied significantly from cell to cell and from each other in the retention of some mink chromosomes. The karyological study of the hybrid lines revealed some cytogenetical specificities of mouse chromosome composition that were general for cells of hybrid lines, but atypical for myeloma parental cells. This data suggest the cells of myeloma parental line to take part in the processes of somatic hybridization and/or selection for mink immunoglobulin production nonrandomly.

[Mechanism of a decrease of inducibility of tyrosine aminotransferase by carcinogenic aminoazo dyes. Absence of direct influence of o-aminoazotoluene on the enzyme molecules]. / K voprosu o mekhanizme snizheniia indutsibel'nosti tirozinaminotransferazy kantserogennymi aminoazokrasiteliami. Otsutstvie priamogo vliianiia o-aminoazotoluola na ferment.

The effect of o-aminoazotoluene (OAT) on the activity of tyrosine aminotransferase (TAT) from mouse liver cytosol under its incubation in the presence of the systems providing for the metabolic activation of the cancerogen (liver microsomes and NADPH2) and dephosphorylation of TAT molecules (light mitochondria and ATP) was studied. It was shown that OAT has neither direct nor indirect (via the phsophorylation--dephosphorylation systems) effect on the activity of TAT. It was concluded that the decrease of TAT induction by hydrocortisone in vivo resulting from injection of OAT to the mice is not due to the direct influence of the cancerogen on the enzyme molecules.