Nariman R. Battulin

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells

Expression of the parental Oct4 and Nanog alleles and DNA methylation of their promoters were studied in a set of Mus musculus embryonic stem (ES) cell/M. caroli splenocyte hybrid cells containing a variable ratio of parental chromosomes 6 and 17. The transcripts of the reactivated splenocyte Oct4 and Nanog genes were revealed in all hybrid cell clones positive for M. caroli chromosomes 6 and 17. We found that 11 CpG sites in the Oct4 promoter were heavily methylated in M. caroli splenocytes (>80%), whereas M. musculus ES cells were essentially unmethylated (<1%). Analysis of the methylation status of the Oct4 promoter in seven hybrid cell clones showed that the splenocyte-derived promoter sequence lost DNA methylation so that its methylation level was comparable with that of the ES cells. Additionally, no preferential de novo methylation was seen in the Oct4 promoters of M. musculus and M. caroli in teratomas developed from two independent hybrid clones. The upstream region of Nanog was heavily methylated in mouse embryonic fibroblasts (66%) and less methylated in M. caroli splenocytes (24%). The Nanog promoter region was completely unmethylated in M. musculus ES cells. We found that both parental alleles of the Nanog gene promoter were essentially unmethylated in five examined hybrid clones. Thus, we have demonstrated that (1) the Oct4 and Nanog genes of splenocytes are activated, and their promoters undergo demethylation in ES cell hybrids; (2) these events are independent of the number and ratio of parental chromosomes carrying these genes.

Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus.

The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.

[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.

Comparison of the 3D organization of sperm and fibroblast genomes using the Hi-C approach

The 3D organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts. The obtained results demonstrated that the 3D genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem (ES) cells. Both A- and B-compartments and topologically associated domains (TADs) are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibited statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome resulted in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. An analysis of interchromosomal contacts in both cell types demonstrated that the large chromosomes showed a tendency to interact with each other more than with the small chromosomes and vice versa. We found that the dependence of the contact probability P(s) on genomic distance for sperm is in a good agreement with the fractal globular folding of chromatin. The similarity of the spatial DNA organization in sperm and somatic cell genomes suggests the stability of the 3D structure of genomes through generations.

Unexpected phenotypic effects of a transgene integration causing a knockout of the endogenous Contactin-5 gene in mice

Contactins (Cntn1-6) are a family of neuronal membrane proteins expressed in the brain. They are required for establishing cell-to-cell contacts between neurons and for the growth and maturation of the axons. In humans, structural genomic variations in the Contactin genes are implicated in neurodevelopmental disorders. In addition, population genetic studies associate Contactins loci with obesity and hypertension. Cntn5 knockout mice were first described in 2003, but showed no gross physiological or behavioral abnormalities (just minor auditory defects). We report a novel Cntn5 knockout mouse line generated by a random transgene integration as an outcome of pronuclear microinjection. Investigation of the transgene integration site revealed that the 6Kbp transgene construct coding for the human granulocyte–macrophage colony-stimulating factor (hGMCSF) replaced 170 Kbp of the Cntn5 gene, including four exons. Reverse transcription PCR analysis of the Cntn5 transcripts in the wild-type and transgenic mouse lines showed that splicing of the transgene leads to a set of chimeric hGMCSF-Cntn5 transcript variants, none of which encode functional Cntn5 protein due to introduction of stop codons. Although Cntn5 knockout animals displayed no abnormalities in behavior, we noted that they were leaner, with less body mass and fat percentage than wild-type animals. Their cardiovascular parameters (heart rate, blood pressure and blood flow speed) were elevated compared to controls. These findings link Cntn5 deficiency to obesity and hypertension.

Morphophysiological effects of insertional mutagenesis of the contactin 5 (Cntn5) gene in transgenic mice

The transgenesis technologies are actively used in different fields of biological studies. The most actively used method to obtain transgenic animals by DNA injection in the zygote pronucleus can be accompanied by the violation of the genes’ function instead of integrating the transgenic construction. In this work, the morphophysiological effects of integrating the transgenic construction providing the production of the human granulocyte–macrophage colony-stimulating factor in the milk of the mouse line (GM9) (the insertion of the construction in the intron of the contactin 5 (Cntn5) gene) were described. It was demonstrated that the insertion of the construction did not result in the Cntn5 knockout. However, the gene transcription in the heart and kidneys of transgenic animals changes compared with wild-type animals, and the spectrum of transcripts also changes, indicating a violation in the Cntn5 gene splicing regulation. It is known from the published data that the Cntn5 polymorphisms are associated with an addiction to obesity and a predisposition to arterial hypertension. We studied the main parameters of the fat metabolism and cardiovascular activity in mice homozygous for the transgene insertion in the Cntn5 gene, heterozygous animals, and wild-type animals. The phenotyping carried out demonstrates that homozygous mice have a smaller body weight than wild-type individuals. And the weight difference between genotypes is determined more by significantly lower fat accumulation than by the lag of mutants in the development of the skeleton and muscles constituting the lean mass. Statistically significant differences in the parameters characterizing the intensity of the blood circulation were found in the studied lines. Homozygous mice exceed wild-type individuals by their arterial pressure values, heart rate, and blood flow rate in the tail vessels. It should be noted that heterozygous individuals have intermediate values between mutants and wild-type according to all the measured morphofunctional parameters.

CRISPR/Cas9, a universal tool for genomic engineering

The CRISPR/Cas9 system was initially described as an element of archeal and bacterial adaptive immunity, but has recently gained much attention in genetic engineering due to its outstanding ability to be programmed to target any genomic loci through a short 20-nucleotide region of single guide RNA (sgRNA). This review deals with some modern applications of the CRISPR/Cas9 system. First, we describe the basic mechanism of the CRISPR/Cas9 DNA recognition and binding with a particular focus on its off-target activity. The CRISPR/Cas9 off-target activity refers to nonspecific recognition of genomic sites that have partial homology with sgRNA. Such activity occasionally results in unwanted mutations throughout the genome. We also note some recent method improvements for enhancing Cas9 specificity or adding new functions to the system. Since the active use of CRISPR/Cas9 is mostly driven by its remarkable potential for gene therapy and genome engineering, the latest CRISPR/Cas9 applications in these areas are also discussed in our review. In particular, the CRISPR/Cas9 system was recently used to control the human immunodeficiency virus (HIV) infection and to repair genetic abnormalities, such as Duchenne muscular dystrophy or retinitis pigmentosa, both in cell cultures and rodent models. The programmable nature of CRISPR/Cas9 facilitates the creation of transgenic organisms through site-specific gene mutations, knock-ins or large chromosomal rearrangements (deletions, inversions and duplications). CRISPR/Cas9 was also found to be especially useful in pronuclear microinjections of farm animals, which has a strong impact on biotechnology. In addition, we reviewed CRISPR/Cas9-based genetic screens, which provide highly efficient identification of new genes and pathways involved in many biological processes. Finally, we discuss the use of gene drivers based on CRISPR/Cas9, which represent a powerful tool for the modification of ecosystems in the nearest future.

Investigation of the spatial genome organization of mouse sperm and fibroblasts by the Hi-C method

The spatial organization of eukaryotic genome plays an important role in the control of gene expression. The new Hi-C method allows investigation of chromosome contact in the three-dimensional architecture of the whole genome; however, it has not yet been applied to study the spatial configuration of the germ cells genome. Here we describe a protocol for production and quality control of Hi-C DNA libraries from mouse sperm cells and fibroblasts. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome.