Generation of four iPSC lines from two siblings with a microdeletion at the CNTN6 gene and intellectual disability.

The human induced pluripotent stem cell (iPSC) lines, ICGi009-A, ICGi009-B, ICGi013-A and ICGi013-B, were generated from skin fibroblasts of two siblings with intellectual disability. Both patients were carriers of CNTN6 gene microdeletion (Kashevarova et al., 2014). iPSC lines have normal karyotype, express pluripotency markers, are able to differentiate in vitro into derivatives of all three germ layers and represent a unique tool to study neurodevelopmental disorders.

[ROLE OF CONTACTINS IN NEUROGENESIS IN HUMAN AND ANIMALS].

Development of central and peripheral nervous system is one of the most complicated processes of embryogenesis. Dendritogenesis is an important component of this process because properties of dendritic branching define input and output signals received by neuron. Moreover, communications between neurons require transition of signal from dendrite of one neuron to axon of another, and this process of signal transduction underlies mechanisms of synaptic plasticity and memory formation. The neural cell adhesion molecules of the immunoglobulin superfamily involved in the control of dendritogenesis. In current review we focus our attention on 6 members of this adhesion molecules family: contactins 1-6. The contactins are proteins that control key events of neurogenesis: adhesion and migration of neuronal cells, orientation of growth of neurites and axons myelination. Functions of contactins are actively studied using model animals that express contactins in central and peripheral nervous system with almost similar to human pattern. Mutations of contactin-encoding genes result in abnormalities of neurogenesis process and development of multiple neurological disorders. Review is devoted to the role of contactin proteins in neurogenesis and nervous system disorders.

[Analysis of chromosome composition in interspecific embryonic stem hybrid cells of mice].

Chromosome complements of twenty hybrid clones obtained by fusion of Mus musculus embryonic stem cells (ESC) and M. caroli splenocytes were studied. Using of double-color in situ hybridization with chromosome- and species-specific probes we were able to detect the parental origin for each chromosome in hybrid cells. Based on parental chromosome ratio, all 20 hybrid clones were separated in some different groups: from the group containing practically tetraploid M. musculus genome with single M. caroli chromosomes to hybrids with dominance of M. caroli chromosome homologues. In 8 hybrid cells clones we observed prevalence of chromosomes originated from ESC in ratio from 5:1 to 3:1. Another hybrid cells clones have either equal (1:1, 1:2) ratio of M. musculus to M. caroli chromosomes or with the prevalence of ESC- (2:1) or splenocyte- (1:2) originated parental chromosome homologues. In 3 hybrid cells clones, we observed preferable segregation of ESC-originated pluripotent chromosomes. This phenomenon was found for the first time and it possibly indicates compensation of the epigenetic differences between parental chromosomes of ESC- and splenocyte-origination.

[FISH-analysis of regional replication in homologous chromosomes in hybrid cells obtained by fusion of embryonic stem cells with somatic cells].

The paper concerns FISH-analysis of regional replication of parental chromosomes 1, 3 and 6 in hybrid cells obtained by fusion between Mus musculus embryonic stem cells (ESC) and M. caroli splenocytes. The data demonstrated that parental chromosomes in the hybrid cells with near-diploid karyotype showed synchronous replication in 70-75% of tested cells that was comparable with diploid ESC and diploid fibroblasts. Synchronous replication of parental chromosomes in hybrid cells with near-triploid karyotype was observed in 46-57% of tested cells. However, it was correct in the case of hybrid cells with three copies of the tested chromosomes whereas the ratio of synchronous replication in triploid cells with two copies was comparable or similar to that in diploid cells. Hybrid cells with near-tetraploid karyotype showed high ratio of asynchronous replication (over 50%) comparable to those tetraploid ESC and tetraploid fibroblasts but significantly distinguished from diploid cells. Thus, most hybrid cells with two copies of tested parental chromosomes showed their synchronous replication. Unfortunately, the FISH-analysis is poor informative when it is used for studying cells with more than two copies of tested chromosomes.

[Fate of parental mitochondria in embryonic stem hybrid cells].

When hybrid cells are created, not only nuclear genomes of parental cells unite but their cytoplasm as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm allowing one to gain insight into the organization of hybrid cell cytoplasm. We analyzed the parental mtDNAs in hybrid cells resulting from fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of the parental mtDNAs in hybrid cells was based on polymorphism among the parental mtDNAs for certain restrictases. We found that intra- and inter-specific ES cell-splenocyte hybrid cells lost entirely or partially mtDNA derived from the somatic partner, whereas ES cell-fibroblast hybrids retained mtDNAs from both parents in similar ratios with a slight bias. The lost of the "somatic" mitochondria by Es-splenocyte hybrids implies non-random segregation of the parental mitochondria as supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from analysis of mtDNA in single cells. Preferential segregation of "somatic" mitochondria does not depend on the differences in sequences of the parental mtDNAs but depends on replicative state of the parental cells.

[Analysis of expression of parental alleles Xist and Gla in interspecific embryonic hybrid cells during induced in vitro inactivation of X-chromosomes].

The results of in situ hybridization with labeled species specific and X-chromosome-specific probes suggest that hybrid cells obtained by fusion of Mus musculus embryonic stem cells (genotype XY) and splenocytes of M. caroli females contain two parental X-chromosomes. In five clones of hybrid cells, differentiation was induced in embryoid bodies in vitro, which was accompanied by inactivation of one of X-chromosomes. We analyzed the expression of Xist and Gla alleles in the embryoid bodies using RT-PCR with an account that expression of locus Xist is one of key events in X-chromosome inactivation, while gene Gla was used as a marker of active X-chromosome. Identification of allele transcripts of loci Xist and Gla was based on restriction polymorphism between M. musculus and M. caroli that we had described. Transcripts of both parental alleles of loci Xist and Gla were present in the embryoid bodies of all studied hybrid clones. No preferential inactivation of M. musculus or M. caroli X-chromosome was found in the tested embryonic hybrid cells despite the initial differences in ontogenetic status between X-chromosomes of embryonic stem cells and splenocytes.

[Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells].

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.

Mapping of 53 loci in American mink (Mustela vison).

Fifty-three genes were mapped in the American mink genome using polymerase chain reaction (PCR)-based analysis of a Chinese hamster-American mink somatic cell hybrid panel. Heterologous primers designed for cat gene mapping were used in this study. Forty-nine of these loci were localized into expected chromosome regions according to Zoo-FISH data, whereas four loci--ALPL, CDC20, ERF-2, and Fc(Mv)23617--were mapped out of expected conserved regions. PCR products amplified with primers corresponding to these four markers were partly sequenced and verified using BLAST. The results showed the homology to be more than 90% between mink and human or cat counterparts. At present, the gene map of American mink has expanded to 127 loci.

[Isolation of ES-like lines of common voles of the genus Microtus from blastocysts and germ cells and as a result of the fusion of somatic cells with mouse embryonic stem cells]. / Poluchenie ES-podobnykh linii obyknovennykh polevok roda Microtus iz blastotsist, germinal'nykh kletok i ot sliianiia somaticheskikh kletok s émbryonal'nymi stvolovymi kletkami myshi.

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F1 Microtus rossiaemeridionalis x M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.

["Chromosome memory" of parental genomes in embryonic hybrid cells]. / "Khromosomnaia pamiat'" roditel'skikh genomov v émbrional'nykh gibridnykh kletkakh.

In the hybrid cells obtained by fusion of embryonic stem cells with adult differentiated cells, homologous chromosomes are in two ontogenetic configurations: pluripotent and differentiated. In order to assess the role of cis- and trans-regulation in the maintenance of these states, we studied a set of clones of hybrid cells of the type embryonic stem cells-splenocytes and used two approaches: segregation of parental chromosomes and comparison of pluripotency of the past hybrid cells and embryonic stem cells. The segregation test showed that the hybrid cells lost only the homologs of the somatic partner and this process was sharply accelerated when the cells were cultivated in nonselective conditions, thus suggesting the full or partial preservation of the initial differences in the organization of parental homologs. The descendants of the former hybrid cells, which had the karyotype similar to that of embryonic stem cells, demonstrated the level of pluripotency, comparable with that of embryonic stem cells despite the long-term effect of trans-acting factors from the somatic partner in the genome of hybrid cells. The data obtained are interpreted in the framework of the concept of "chromosome memory", in the maintenance of which the key role is played by cis-regulatory factors.

[Embryonal cell hybrids: new opportunities for studying pluripotency and reprogramming of the differentiated cell chromosomes]. / Embrional'nye gibridnye kletki: novye vozmozhnosti v izuchenii pliuripotentnosti i reprogrammirovaniia khromosom diffenetsirovannykh kletok.

Here we study the properties of cell hybrids produced by the fusion of embryonal stem cells and differentiated ones. During in vitro cultivation, such hybrids predominantly lose the somatic partner chromosomes, although the loss of the embryonic partner autosomes 1, 9, 11, 12, 15, 16, 18, and 19 is also common in the clones; i.e., this is a bidirectional process. The use of a selective media allows the isolation of the clones, with the embryonal X chromosome replaced by the somatic genome homolog. The cell hybrids with a near-diploid chromosome set preserve the high-level pluripotency properties of the embryonal partner including the capacity to form chimeras after their introduction in the blastocoel. An investigation of the chimeric animals demonstrated a reprogramming of the "somatic" X chromosome in the course of development. The prospective identification of the chromosomes involved in the maintenance of pluripotency and studies of its cis- and trans-regulation in the cell hybrid genome are discussed.

Genetic modification of mammalian genome at chromosome level.

The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs) provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.

[Torsional tension in metaphase chromosome DNA]. / Torsionnoe napriazhenie v DNK metafaznykh khromosom.

The torsional tension in DNA of isolated metaphase chromosomes from murine fibroblasts was measured by the microfluorescent method. The method is based on the ability of a fluorescent dye ethidium bromide to compensate for the negative torsional tension in topologically closed DNA by intercalation between DNA base pairs. The value of the relative twist difference delta Tw/Tw = -0.1 was found in a bulk (about 3/4) of unconstrained chromosomal DNA. In interphase nuclei, the torsionally stressed DNA comprises about 15%, with value of delta Tw/Tw = -0.075. We suppose that the tension in chromosomal DNA was created in the prophase stage of mitosis by condensines, the drivers of chromosomal condensation.

In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes.

Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype.

Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells.

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.

Novel strategies for eutherian x marsupial somatic cell hybrids: mapping the genome of Monodelphis domestica.

Two hundred thirty-seven independent somatic cell hybrids have been obtained between opossum (Monodelphis domestica) splenocytes, bone marrow cells, or primary fibroblasts, and HPRT-deficient or TK-deficient Chinese hamster, mouse, American mink, or common vole fibroblast lines. Because extreme segregation and fragmentation of marsupial chromosomes commonly occurs in eutherian x marsupial somatic cells hybrids, we developed a rapid primary screening method that enables the identification of primary clones containing a large amount of opossum DNA 20-25 d after fusion. This method, which depends on in situ hybridization of biotin-labeled total opossum DNA on interphase nuclei of hybrid cells fixed on the bottom of microwell plates, was used to screen the 237 hybrid clones; 52 of them had a substantial amount of opossum DNA. G-banding and in situ hybridization of biotin-labeled total opossum DNA on metaphase spreads of the clones enabled identification of 17 hybrid clones containing from two to seven intact chromosomes of M. domestica on the background of Chinese hamster or vole chromosomes. The hybrid clones with intact opossum chromosomes are used in a panel constructed for mapping the opossum genome. Initial mapping results from these clones have led to the tentative assignment of GPI and GOT1 to chromosome 1; 6PGD to chromosome 4; LDHA to chromosome 5; LDHB to chromosome 8; and PGK and G6PD to the X chromosome. On the basis of indirect evidence we also tentatively assigned HPRT to the X chromosome and TK to chromosome 5 of M. domestica. These are the first tentative chromosomal assignments by any technique for this species.

Chromosomal and regional localization of the loci for IGKC, IGGC, ALDB, HOXB, GPT, and PRNP in the American mink (Mustela vison): comparisons with human and mouse.

Chromosomal localization of the genes for gamma- and kappa-immunoglobulins (IGGC and IGKC, respectively), aldolase B (ALDB), prion protein (PRNP), homeo box B (HOXB), and glutamate pyruvate transaminase (GPT) were determined with the use of mink-rodent hybrid cells. Analysis of segregation of the mink markers and chromosomes in these hybrid cells allowed us to assign the gene for HOXB to Chromosome (Chr) 8, IGGC to Chr 10, PRNP and IGKC to Chr 11, ALDB to Chr 12, and GPT to Chr 14 in mink. Furthermore, using a set of mink-mouse hybrid cells carrying fragments of mink Chr 8 of different sizes, we assigned the gene for HOXB to the pter-p26 region of the short arm of Chr 8. Comparative mapping of the genes of mink, human, and mouse, as well as other mammalian species, demonstrated that the mink genes HOXB, PRNP, ALDB, and IGGC are members of a conserved region shared by many mammalian species in common; the IGKC gene is a member of a conserved region common to carnivores and primates, not rodents; the GPT gene is a member of a syntenic gene group probably unique to the Mustelidae family or carnivores.