Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.     

“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.     

Chromosome composition of interspecies hybrid embryonic stem cells in mice

Chromosome complements of 20 hybrid clones obtained by fusing Mus musculus embryonic stem cells (ESCs) and Mus caroli splenocytes were studied. The use of two-color fluorescence hybridization in situ with chromosome- and species-specific probes has allowed us to reliably reveal the parental origin of homologs of any chromosome in hybrid cells. Depending on the ratio of parental chromosome homologs, all 20 hybrid clones were separated in several groups ranging from the clones that contain cells that are nearly tetraploid with two diploid sets of M. musculus and single M. caroli chromosomes to clones with a marked predominance of the M. caroli chromosome. In eight hybrid cell clones, we observed the pronounced prevalence of chromosomes of the pluripotent partner over chromosomes of the somatic partner in a ratio of 5: 1 to 3: 1. In other hybrid cell clones, the ratio of M. musculus to M. caroli chromosomes was either equal (1: 1; 2: 2) or with the prevalence of the pluripotent (2: 1) or differentiated (1: 2) partner. In three hybrid cell clones, for the first time, we observed the predominant segregation of ESC-derived pluripotent chromosomes. This might indicate the compensation for the epigenetic differences between parental chromosomes of the ESC and splenocyte origin.     

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.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 151–158.Original Russian Text Copyright © 2005 by Pristyazhnyuk, Temirova, Menzorov, Kruglova, Matveeva, Serov.     

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.     

Assessment of vegetation cover by lichen-indication methods (by the example of the north of Central Siberia)

The vegetation cover in the north of Central Siberia has been assessed from lichenological data. The approaches and methods of lichen-based indication of the state of vegetation cover have been developed with the application of the data on diversity of lichens and lichen synusias, their projective cover and morphological deviations. The species of lichens and synusias that can be used for indication purposes have been defined. The changes in the characteristics of the lichen component in vegetation cover under the increasing chemical air pollution caused by industrial plants have been examined. The early deviations are indicated by morphological changes of lichen thalli, species depletion, and diversity of lichen synusias. Practical recommendations on lichen indication of the vegetation cover in the north of Siberia have been given.     

Development and gene expression of nuclear transplants generated by transplantation of cultured cell nuclei into non-enucleated eggs in the medaka Oryzias latipes

To develop nuclear transplantation techniques for the medaka Oryzias latipes, nuclei of cultured cells from transgenic fish were transplanted into unfertilized eggs of the orange-red variety of O. latipes, without enucleation, in two experimental series. In the first experimental series, fibroblast cells cultured from the adult caudal fin were used as donors, which carried the green fluorescent protein (GFP) gene driven by the promoter of the medaka elongation factor 1alpha-A gene. Wild-type body color was another donor genetic marker used in this experimental series. In the second experimental series, cells cultured from 6-day-old embryos were used as donors, which carried the GFP genetic marker driven by the promoter of the medaka beta-actin gene. From more than 1000 eggs transplanted in each experiment, a considerable number of nuclear transplants developed to various embryonic stages showing stage- and tissue-specific expression of the donor genetic markers, although the expression was mosaic in many cases. Three and six of the transplanted eggs in the first and second experimental series (0.3 and 0.5%, respectively) hatched, and the hatchlings expressing the genetic markers survived for up to 3 weeks. The chromosome number varied among cells in a single transplant embryo. The results obtained in these experiments may help future cloning efforts in fish.     

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

The paper deals with the FISH analysis of the regional replication of homologue of chromosomes 1, 3, and 6 in hybrid cells obtained by the fusion of Mus musculus embryonic stem cells (ESCs) and somatic cells—M. caroli splenocytes. The obtained data showed that, in hybrid cells with near-diploid karyotypes, the parental chromosomes were replicated synchronously in 70–75% of tested cells, similar to in diploid ESCs and diploid fibroblasts. In hybrid cells with near-triploid karyotypes, the asynchronous replication of the parental chromosomes increased to 46–57% of tested cells. However, this is true for hybrid cells with three copies of tested chromosomes, whereas, in triploid cells with two copies, the level of the homolog synchronous replication was close to that of diploid cells. In hybrid cells with near-tetraploid karyotypes, the level of asynchronous replication was observed in more than 50% of cells, which is comparable with the level in tetraploid ESCs and tetraploid fibroblasts. Thus, in hybrid cells with no more than two copies of an individual chromosome, the synchronous replication of homologue that initially had different levels of differentiation and parameters of replications was observed. However, the information value of the method of in situ hybridization on interphase nuclei changes significantly with an increase in the number of copies of individual chromosomes and thereby restricts possibilities of this approach for evaluation of synchronous homolog replication in hybrid cells.     

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129 × BALB (1B), C57BL × 1B, and DD × 1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9 %, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0 % of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XX-cell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.     

Unequal segregation of parental chromosomes in embryonic stem cell hybrids

Chromosome segregation was studied in 14 intra- and 20 inter-specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species-specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES x ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES-splenocyte cell hybrids, but lower in ES-fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype.