Embryonic stem cells derived from morulae,inner cell mass,and blastocysts of mink: Comparisons of their pluripotencies

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley-Liss, Inc.     

“Chromosome Memory” of Parental Genomes in Embryonic Hybrid Cells

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 bycis-regulatory factors.     

Monitoring of the chitinolytic microbial complex of the phylloplane

A comprehensive study of chitinolytic microbial complexes of the phylloplane from cultured and forest plants has been conducted. An increase of the number and biomass of metabolically active cells of the representatives of the domain Bacteria and a decrease in fungal biomass in the experimental microcosms have been shown to occur after the introduction of chitin. The characteristic features of the taxonomic structure of metabolically active chitinolytic complexes of the phylloplane of the plants studied have been elucidated. Representatives of the phyla Proteobacteria, Bacteroidetes, and Verrucomicrobia have been shown to play important roles in the chitinolytic complexes of green leaf samples, while mycelial actinobacteria of the phylum Actinobacyteria played a similar role in needles of coniferous trees. A collection of chitinolytic microorganism cultures isolated from the phylloplane of different plant species has been created.     

Immunocytological analysis of meiotic recombination in the American mink (Mustela vison)

Using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites along synaptonemal complexes, we estimated the total length of the genetic map, the recombination rate and crossover distribution in the American mink ( Mustela vison ). We prepared spreads from 130 spermatocytes of five male minks and mapped 3320 MLH1 foci along 1820 bivalents. The total recombination length of the male mink genome, based on the mean number of MLH1 foci for all chromosomes, was 1327 cM. The overall recombination rate was estimated to be 0.48 cM/Mb. In all bivalents, we observed prominent peaks of MLH1 foci near the distal ends and a paucity of them near the centromeres. This indicates that genes located at proximal regions of the chromosomes should display much tighter genetic linkage than physically equidistant markers located near the telomeres.     

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. Mol. Reprod. Dev. 50:128–138, 1998. © 1998 Wiley-Liss, Inc.     

Microbiomes of Virgin Soils of Southern Vietnam Tropical Forests

Microbiology - Numbers of bacterial, archaeal, and fungal ribosomal gene copies and the taxonomic structure of prokaryotic communities in virgin tropical soils under weakly impacted monsoon forests...     

Effect of genetic factors on the rate of embryonic growth in mink]

A comparative analysis of the growth rate of embryos of two groups of minks differing in the coat colour was carried out. For comparison, 10 development stages with progressively more complicated morphology were taken. It has been demonstrated that the difference in the emmbryo growth rate between the standard and "sapphire" colour forms appear at the "pre-fetal" and, especially, "fetal" period of development.     

Isolation of ES-Like Lines of Common Voles of the Genus Microtus from Blastocysts and Germ Cells and as a Result of Fusion of Somatic Cells with Mouse Embryonic Stem Cells

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 × 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.