Knockout of the Tnfa Gene Decreases Influenza Virus-Induced Histological Reactions in Laboratory Mice.

Tumor necrosis factor alpha (TNF-α) is a cytokine that is responsible for many processes associated with immune response and inflammation. It is involved in the development of an antiviral response to many virus infections. This factor was shown to be activated in influenza A virus infection, which enhances production of other cytokines. The overexpression of these cytokines can lead to a cytokine storm. To study the role of TNF-α in the development of pathologies associated with viral infection, we generated a Tnfa knockout mouse strain. We demonstrated that these mice were characterized by a significant increase in the number of viral genomes compared to that in the parental strain, but the amount of live virus did not differ. A histopathology of the lungs in the genetically modified animals was significantly lower in terms of interalveolar septal infiltration. The generated model may be used to further study pathological processes in viral infections.

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.

Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique.

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells. High-throughput sequencing of barcodes from successfully reprogrammed cells revealed a significant number of barcodes from related cells. We developed a computer model, according to which a probability of synchronous reprogramming of sister cells equals 10-30%. We conclude that the reprogramming success is pre-established in some particular cells and, being a heritable trait, can be maintained through cell division. Thus, reprogramming progresses in a deterministic manner, at least at the level of cell lineages.

Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice.

AIM: To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells. METHODS: Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS: The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts. CONCLUSION: Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.

A 3,387 bp 5'-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice.

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 µg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.

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.

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.

Dominant manifestation of pluripotency in embryonic stem cell hybrids with various numbers of somatic chromosomes.

Developmental potential was assessed in 8 intra-specific and 20 inter-specific hybrid clones obtained by fusion of embryonic stem (ES) cells with either splenocytes or fetal fibroblasts. Number of chromosomes derived from ES cells in these hybrid clones was stable while contribution of somatic partner varied from single chromosomes to complete complement. This allowed us to compare pluripotency of the hybrid cells with various numbers of somatic chromosomes. Three criteria were used for the assessment: (i) expression of Oct-4 and Nanog genes; (ii) analyses of teratomas generated by subcutaneous injections of the tested cells into immunodeficient mice; (iii) contribution of the hybrid cells in chimeras generated by injection of the tested cells into C57BL blastocysts. All tested hybrid clones showed expression of Oct-4 and Nanog at level comparable to ES cells. Histological and immunofluorescent analyses demonstrated that most teratomas formed from the hybrid cells with different number of somatic chromosomes contained derivatives of three embryonic layers. Tested hybrid clones make similar contribution in various tissues of chimeras in spite of significant differences in the number of somatic chromosomes they contained. The data indicate that pluripotency is manifested as a dominant trait in the ES hybrid cells and does not depend substantially on the number of somatic chromosomes. The latter suggests that the developmental potential derived from ES cells is maintained in ES-somatic cell hybrids by cis-manner and is rather resistant to trans-acting factors emitted from the somatic one.