Sorex araneus and Sorex granarius are sibling species within the Sorex araneus group with karyotypes composed of almost identical chromosome arms. S. granarius has a largely acrocentric karyotype, while, in S. araneus, various of these acrocentrics have combined together by Robertsonian (Rb) fusions to form metacentrics, with the numbers and types of metacentrics differing between chromosomal races. Our studies on telomeric sequences in S. araneus and S. granarius revealed differences between chromosomes and between species. In S. araneus (the Novosibirsk race), hybridization signals were present on the telomeres of all the chromosomes after FISH with a PCR-generated telomeric probe. In addition, hybridization signals were observed at high frequencies in the pericentric regions of some but not all metacentrics formed by Rb fusion. There were fewer signals on those metacentrics formed earlier in the evolution of S. araneus. This suggests that S. araneus chromosomes retain at least some telomeric repeats during Rb fusion, but that these repeats are lost or modified over time. These results are critical for the interpretation of the well-studied hybrid zones between chromosomal races of S. araneus, given that Rb fission has been postulated in such hybrid zones and that the likelihood of Rb fission will relate to presence/absence of telomeric sequences at the centromeres of metacentrics. In S. granarius, there were strong signals at the proximal (centromeric) telomeres of the acrocentrics after FISH with a DNA telomeric probe. FISH with a PNA telomeric probe on S. granarius acrocentrics showed that the proximal telomeres were 213 kb on average, while the length of the distal telomeres was 3.8 kb on average. Two-colour FISH, using a telomeric DNA probe and a microdissected probe generated from the pericentric regions of the S. granarius chromosomes a and b, revealed regions on distinct chromatin fibres where telomeric and microdissected probes were colocalized or localized sequentially. The proximal telomeres of S. granarius are highly unusual both in their large size and their heterogeneous structure relative to the telomeres of other mammals.
The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.
Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and largescale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps ([ 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a Elena V. Grigor'eva and Tuyana B. Malankhanova contributed equally to this work.
Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types.
Two closely related shrew species, Sorex granarius and Sorex araneus, in which Robertsonian rearrangements have played a primary role in karyotype evolution, present very distinct telomere length patterns. S. granarius displays hyperlong telomeres specifically associated with the short arms of acrocentrics, whereas telomere lengths in S. araneus are rather short and homogenous. Using a combined approach of chromosome and fibre FISH, modified Q-FISH, 3D-FISH, Ag-NOR staining and TRF analysis, we carried out a comparative analysis of telomeric repeats and rDNA distribution on chromosome ends of Sorex granarius. Our results show that rDNA sequences forming active nuclear organizing regions are interspersed with the long telomere tracts of all short arms of acrocentrics. These observations suggest that the major rearrangements that gave rise to today's karyotype in S. granarius were accompanied by a profound reorganization of chromosome ends, which comprised extensive amplification of telomeric and rDNA repeats on the short arms of acrocentrics and finally contributed to the stabilization of telomeres. This is the first time that such telomeric structures have been observed in any mammalian species.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.