Creation of transgenic mice susceptible to coronaviruses: a platform for studying viral pathogenesis and testing vaccines.

Over the past 20 years, coronaviruses have caused three epidemics: SARS-CoV, MERS-CoV, and SARS-CoV2, with the f irst two having a very high lethality of about 10 and 26 %, respectively. The last outbreak of coronavirus infection caused by SARS-CoV2 in 2019 in China has swept the entire planet and is still spreading. The source of these viruses in humans are animals: bats, Himalayan civets, and camels. The genomes of MERS-CoV, SARS-CoV and SARS-CoV2 are highly similar. It has been established that coronavirus infection (SARS-CoV and SARS-CoV2) occurs through the viral protein S interaction with the lung epithelium - angiotensin-converting enzyme receptor 2 (ACE2) - due to which the virus enters the cells. The most attractive model for studying the development of these diseases is a laboratory mouse, which, however, is resistant to coronavirus infection. The resistance is explained by the difference in the amino acid composition of mouse Ace2 and human ACE2 proteins. Therefore, to create mice susceptible to SARS- CoV and SARS-CoV2 coronaviruses, the human ACE2 gene is transferred into their genome. The exogenous DNA of the constructs is inserted into the recipient genome randomly and with a varying number of copies. Based on this technology, lines of transgenic mice susceptible to intranasal coronavirus infection have been created. In addition, the use of the technology of targeted genome modif ication using CRISPR/Cas9 made it possible to create lines of transgenic animals with the insertion of the human ACE2 gene under the control of the endogenous murine Ace2 gene promoter. This "humanization" of the Ace2 gene makes it possible to obtain animals susceptible to infection with coronaviruses. Thus, transgenic animals that simulate coronavirus infections and are potential platforms for testing vaccines have now been created.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Generation of iPS cell line (ICGi040-A) from skin fibroblasts of a patient with ring small supernumerary marker chromosome 4.

Human induced pluripotent stem cell (iPSC) line, ICGi040-A, was obtained from skin fibroblasts derived from a male patient with mosaic ring small supernumerary marker chromosome 4 (sSMS(4)) and infertility. ICGi040-A cells have karyotype 47,XY,+r(4) in 97% of cells and express a set of pluripotent markers, as well as are able to differentiate in vitro into derivatives of all three embryonic germ layers.

Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming.

Human ring chromosomes are often unstable during mitosis, and daughter cells can be partially or completely aneuploid. We studied the mitotic stability of four ring chromosomes, 8, 13, 18, and 22, in long-term cultures of skin fibroblasts and induced pluripotent stem cells (iPSCs) by GTG karyotyping and aCGH. Ring chromosome loss and secondary aberrations were observed in all fibroblast cultures except for r(18). We found monosomy, fragmentation, and translocation of indexed chromosomes. In iPSCs, aCGH revealed striking differences in mitotic stability both between iPSC lines with different rings and, in some cases, between cell lines with the same ring chromosome. We registered the spontaneous rescue of karyotype 46,XY,r(8) to 46,XY in all six iPSC lines through ring chromosome loss and intact homologue duplication with isoUPD(8)pat occurrence, as proven by SNP genotype distribution analysis. In iPSCs with other ring chromosomes, karyotype correction was not observed. Our results suggest that spontaneous correction of the karyotype with ring chromosomes in iPSCs is not universal and that pluripotency is compatible with a wide range of derivative karyotypes. We conclude that marked variability in the frequency of secondary rearrangements exists in both fibroblast and iPSC cultures, expanding the clinical significance of the constitutional ring chromosome.

Establishment of an induced pluripotent stem cell line (ICGi026-A) from peripheral blood mononuclear cells of a patient with fragile X syndrome.

Expansion over 200 CGG repeats in FMR1 gene causes inherited intellectual disability or autism spectrum disorder named as fragile X syndrome. Despite the known cause fragile X syndrome pathogenesis has not been specified yet. The ICGi026-A iPSCs line was obtained by the reprogramming of the peripheral blood mononuclear cells from a 9-year-old boy with fragile X syndrome. The ICGi026-A iPSCs expressed pluripotency markers, had a normal male karyotype (46, XY) and had the capacity to in vivo differentiate into the cells of three germ layers.

Generation of iPSC line ICGi024-A from human skin fibroblasts of a patient with ring chromosome 18.

Ring chromosome 18 is a rare chromosomal disorders that usually originate de novo and correlate with clinical manifestation: developmental delay as well as microcephaly, brain and ocular malformations, hypotonia and skeletal abnormalities. We generate iPSC clonal cell line ICGi024-A with pluripotency properties which were demonstrated in vitro by three germ layer differentiation capacity. ICGi024-A can be used for disease modeling and fundamental investigation of ring chromosome instability.

Establishment of an induced pluripotent stem cell line (ICGi025-A) from fibroblasts of a patient with 46,XY,r(8)/45,XY,-8 mosaicism.

Ring chromosomes are structural aberrations commonly associated with disease phenotype. We consider necessary to create the iPSCs with a ring chromosome 8, which can be used for disease modeling and related research. The ICGi025-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a 1-year-old boy with 46,XY,r(8)/45,XY,-8 mosaicism, developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, moderate proximal muscle weakness, feeding problems, and motor alalia. The iPSCs had expression of the pluripotency-associated markers. In vitro differentiated cells expressed the markers of the cells of three germ layers. That data allowed us to conclude that ICGi025-A cells were pluripotent.

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.

Generation of the induced pluripotent stem cell line, ICAGi002-A, from unaffected carrier megabase scaled duplication involving the CNTN6 gene.

The 3p26.3 microduplication involving the CNTN6 gene cause developmental delay and the intellectual disability. However, the incomplete penetrance is described for this copy number variation (CNV). Here we describe ICAGi002-A line, which is supposed to use as a model for studying of the penetrance of the CNV in 3p26.3. The ICAGi002-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a healthy donor with 3p26.3 microduplication involving the CNTN6 gene. The ICAGi002-A cells was pluripotent as it was shown by the expression of the pluripotency-associated markers and in vitro differentiation into the cells of three germ layers.

Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication.

Skin fibroblasts from a patient with developmental delay and chromosome 2p25.3 deletion syndrome were reprogrammed into induced pluripotent stem cells (iPSCs) and the clonal stem cell line ICAGi001-A (iTAF9-11) was established. ICAGi001-A pluripotency was demonstrated in vitro by three germ layer differentiation capacity. This line is a good model for studying of the developmental delay and brain disorder.

Induced pluripotent stem cell line, IMGTi003-A, derived from skin fibroblasts of an intellectually disabled patient with ring chromosome 13.

Skin fibroblasts from a patient with neurodevelopmental and speech delay, anxiety disorder, macrocephaly, microorchidism, multiple anomalies of internal organs and ring chromosome 13 were reprogrammed into induced pluripotent stem cells (iPSCs) to generate a clonal stem cell line IMGTi003-A (iTAF6-6). IMGTi003-A pluripotency was demonstrated by three germ layer differentiation capacity in vitro, and this cell line had a mosaic karyotype with 46,XY,r(13) as a predominant cell subpopulation. IMGTi003-A line is a good model for studying of the mitotic instability of the ring chromosome 13.

Generation of two iPSC lines (IMGTi001-A and IMGTi001-B) from human skin fibroblasts with ring chromosome 22.

Skin fibroblasts from a patient with intellectual disability and ring chromosome 22 were reprogrammed into induced pluripotent stem cells (iPSCs) to establish a clonal stem cell lines, IMGTi001-A (iTAF5-29) and IMGTi001-B (iTAF5-32). Because of ring chromosome mitotic instability these cell lines show mosaic karyotypes with 46,XX,r(22) in >83% cells, 45,XX,-22 as minor class and sporadically cells with other karyotypes. Differentiation in derivatives of all three germ layers was shown in teratoma assay for IMGTi001-A, and in embryoid bodies for both cell lines. To our knowledge, human iPSC lines with ring chromosome are described for the first time.

Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

[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.

[Reprogramming of somatic cells. Problems and solutions].

An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.

Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 µg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 µg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

[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.

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.