scholarly journals Sleeping Beauty Transposon-Based Phenotypic Analysis of Mice: Lack of Arpc3 Results in Defective Trophoblast Outgrowth

2006 ◽  
Vol 26 (16) ◽  
pp. 6185-6196 ◽  
Author(s):  
Kojiro Yae ◽  
Vincent W. Keng ◽  
Masato Koike ◽  
Kosuke Yusa ◽  
Michiyoshi Kouno ◽  
...  
Keyword(s):  
Microscopic Analysis ◽  
Blastocyst Stage ◽  
Sleeping Beauty ◽  
Cell Periphery ◽  
Phenotypic Analysis ◽  
Electron Microscopic ◽  
Sleeping Beauty Transposon ◽  
Compound Heterozygotes

ABSTRACT The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.

scholarly journals DSS1 and ssDNA regulate oligomerization of BRCA2

2020 ◽  
Vol 48 (14) ◽  
pp. 7818-7833 ◽  
Author(s):  
Hang Phuong Le ◽  
Xiaoyan Ma ◽  
Jorge Vaquero ◽  
Megan Brinkmeyer ◽  
Fei Guo ◽  
...  
Keyword(s):  
Replication Fork ◽  
Microscopic Analysis ◽  
Filament Formation ◽  
Electron Microscopic ◽  
Microscopic Imaging ◽  
Cellular Events ◽  
Self Association ◽  
Oligomeric Forms

Abstract The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.

356 SLEEPING BEAUTY TRANSGENESIS IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 266 ◽  
Author(s):  
W. Garrels ◽  
T. R. Talluri ◽  
R. Bevacqua ◽  
A. Alessio ◽  
A. Fili ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Small Animal ◽  
Blastocyst Stage ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Phenotypic Analysis ◽  
Helper Plasmid ◽  
Specific Expression ◽  
Spontaneous Bleeding

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.Financial support of DFG (Ku 1586/3-1), UNRC, CONICET and Agencia Nacional de Promoción Científica y Tecnológica de la Argentina (ANPCyT) is gratefully acknowledged.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

Abstract WMP31: Dedifferentiation of Smooth Muscle Cells and its Potential Contribution to Pathogenesis of Intracranial Aneurysms

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Mieko Oka ◽  
Nobuhiko Ohno ◽  
Takakazu Kawamata ◽  
Tomohiro Aoki
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Inflammatory Responses ◽  
Muscle Cells ◽  
Inflammatory Factors ◽  
Potential Contribution ◽  
Electron Microscopic

Introduction: Intracranial aneurysm (IA) affects 1 to 5 % in general public and becomes the primary cause of subarachnoid hemorrhage, the most severe form of stroke. However, currently, no drug therapy is available for IAs to prevent progression and rupture of lesions. Elucidation of mechanisms underlying the disease is thus mandatory. Considering the important role of vascular smooth muscle cells (SMCs) in the maintenance of stiffness of arterial walls and also in the pathogenesis of atherosclerosis via mediating inflammatory responses, we in the present study analyzed morphological or phenotypical changes of SMCs during the disease development in the lesions. Methods: We subjected rats to an IA model in which lesions are induced by increase of hemodynamic force loading on intracranial arterial bifurcations and performed histopathological analyses of induced lesions including the electron microscopic examination. We then immunostained specimens from induced lesions to explore factors responsible for dedifferentiation or migration of SMCs. In vitro study was also done to examine effect of some candidate factors on dedifferentiation or migration of cultured SMCs. Results: We first found the accumulation of SMCs underneath the endothelial cell layer mainly at the neck portion of the lesion. These cells was positive for the embryonic form of myosin heavy chain, a marker for the dedifferentiated SMCs, and the expression of pro-inflammatory factors like TNF-α. In immunostaining to explore the potential factor regulating the dedifferentiation of SMCs, we found that Platelet-derived growth factor-BB (PDGF-BB) was expressed in endothelial cells at the neck portion of IA walls. Consistently, recombinant PDGF-BB could promote the dedifferentiate of SMCs and chemo-attracted them in in vitro. Finally, in the stenosis model of the carotid artery, PDGF-BB expression was induced in endothelial cells in which high wall shear stress was loaded and the dedifferentiation of SMCs occurred there. Conclusions: The findings from the present study imply the role of dedifferentiated SMCs partially recruited by PDGF-BB from endothelial cells in the formation of inflammatory microenvironment at the neck portion of IA walls, leading to the progression of the disease.

scholarly journals Silver Nanoparticles Embedded in Natural Rubber Films: Synthesis, Characterization, and Evaluation ofIn VitroToxicity

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Caroline S. Danna ◽  
Dalita G. S. M. Cavalcante ◽  
Andressa S. Gomes ◽  
Leandra E. Kerche-Silva ◽  
Eidi Yoshihara ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Natural Rubber ◽  
Chinese Hamster ◽  
Cell Lineage ◽  
Microscopic Analysis ◽  
Visible Spectrum ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Atomic Force

Natural rubber (NR) films can reduce silver metal ions forming embedded metal nanoparticles, a process that could be described as green synthesis. The NR films acting as a reactor generate and incorporate silver nanoparticles (AgNPs). Organic acids and amino acids play a crucial role in the formation of AgNPs. The plasmon extinction obtained in the UV-visible spectrum shows the presence of nanoparticles in the film after dipping the NR film into a solution of silver nitrate at 80°C. Electron microscopic analysis confirms the presence of AgNPs in the NR film and characterization by atomic force microscopy shows a change in the roughness of the NR film with AgNPs. In addition, our preliminary results fromin vitrotoxicity studies (MTT and comet assays) of the NR films and NR films with silver nanoparticles (NR/Ag) show that they are not toxic to cell lineage CHO-K1 (cells from the ovary of a Chinese hamster), an important result for potential medical applications.

scholarly journals Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice

Reproduction ◽  
2010 ◽  
Vol 139 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Samu Myllymaa ◽  
Arja Pasternack ◽  
David G Mottershead ◽  
Matti Poutanen ◽  
Minna M Pulkki ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Microscopic Analysis ◽  
Corpora Lutea ◽  
Electron Microscopic ◽  
Oocyte Growth ◽  
Long Term Study ◽  
Ovarian Folliculogenesis

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain–Fc fusion protein (BMPR2ecd–Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd–Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd–Fc during the postnatal days 4–12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd–Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4–28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd–Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

scholarly journals Candida albicans forms biofilms on the vaginal mucosa

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3635-3644 ◽  
Author(s):  
M. M. Harriott ◽  
E. A. Lilly ◽  
T. E. Rodriguez ◽  
P. L. Fidel ◽  
M. C. Noverr
Keyword(s):  
Biofilm Formation ◽  
Ex Vivo ◽  
Microscopic Analysis ◽  
Vaginal Mucosa ◽  
First Time ◽  
Established Role ◽  
Type Strains

Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).

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