scholarly journals A transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanisms

2021 ◽  
Vol 25 (1) ◽  
pp. 117-124
Author(s):  
I. V. Grishchenko ◽  
A. A. Tulupov ◽  
Y. M. Rymareva ◽  
E. D. Petrovskiy ◽  
A. A. Savelov ◽  
...  
Keyword(s):  
Cell Line ◽  
Fragile X Syndrome ◽  
Cell Lines ◽  
Fragile X ◽  
Secondary Structures ◽  
Repeat Expansion ◽  
Dna Metabolism ◽  
Repeat Instability ◽  
Transgenic Cell ◽  
Dna Secondary Structures

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully conf irmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR­-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantif ication was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for f inding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantif ication of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.

scholarly journals Inhibitors of Histone Deacetylases Are Weak Activators of the FMR1 Gene in Fragile X Syndrome Cell Lines

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Alexander A. Dolskiy ◽  
Vladimir O. Pustylnyak ◽  
Andrey A. Yarushkin ◽  
Natalya A. Lemskaya ◽  
Dmitry V. Yudkin
Keyword(s):  
Fragile X Syndrome ◽  
Cell Lines ◽  
Histone Deacetylases ◽  
Neuronal Development ◽  
Fragile X ◽  
Repeat Expansion ◽  
Symptomatic Therapy ◽  
Cgg Repeat ◽  
Clinic Practice ◽  
Patient Cell

Fragile X syndrome is the most common cause of inherited intellectual disability in humans. It is a result of CGG repeat expansion in the 5′ untranslated region (5′ UTR) of the FMR1 gene. This gene encodes the FMRP protein that is involved in neuronal development. Repeat expansion leads to heterochromatinization of the promoter, gene silencing, and the subsequent absence of FMRP. To date, there is no specific therapy for the syndrome. All treatments in clinic practice provide symptomatic therapy. The development of drug therapy for Fragile X syndrome treatment is connected with the search for inhibitors of enzymes that are responsible for heterochromatinization. Here, we report a weak transcriptional activity of the FMR1 gene and the absence of FMRP protein after Fragile X syndrome cell lines treatment with two FDA approved inhibitors of histone deacetylases, romidepsin and vorinostat. We demonstrate that romidepsin, an inhibitor of class I histone deacetylases, does not activate FMR1 expression in patient cell cultures, whereas vorinostat, an inhibitor of classes I and II histone deacetylases, activates a low level of FMR1 expression in some patient cell lines.

scholarly journals Spatially coordinated heterochromatinization of distal short tandem repeats in fragile X syndrome

2021 ◽  
Author(s):  
Linda Zhou ◽  
Chunmin Ge ◽  
Thomas Malachowski ◽  
Ji Hun Kim ◽  
Keerthivasan Raanin Chandradoss ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Fragile X ◽  
Repeat Expansion ◽  
Genome Wide ◽  
A Genome ◽  
In Trans ◽  
Surveillance Mechanism ◽  
Short Tandem

AbstractShort tandem repeat (STR) instability is causally linked to pathologic transcriptional silencing in a subset of repeat expansion disorders. In fragile X syndrome (FXS), instability of a single CGG STR tract is thought to repress FMR1 via local DNA methylation. Here, we report the acquisition of more than ten Megabase-sized H3K9me3 domains in FXS, including a 5-8 Megabase block around FMR1. Distal H3K9me3 domains encompass synaptic genes with STR instability, and spatially co-localize in trans concurrently with FMR1 CGG expansion and the dissolution of TADs. CRISPR engineering of mutation-length FMR1 CGG to normal-length preserves heterochromatin, whereas cut-out to pre-mutation-length attenuates a subset of H3K9me3 domains. Overexpression of a pre-mutation-length CGG de-represses both FMR1 and distal heterochromatinized genes, indicating that long-range H3K9me3-mediated silencing is exquisitely sensitive to STR length. Together, our data uncover a genome-wide surveillance mechanism by which STR tracts spatially communicate over vast distances to heterochromatinize the pathologically unstable genome in FXS.One-Sentence SummaryHeterochromatinization of distal synaptic genes with repeat instability in fragile X is reversible by overexpression of a pre-mutation length CGG tract.

scholarly journals Urine-Derived Epithelial Cell Lines: A New Tool to Model Fragile X Syndrome (FXS)

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2240
Author(s):  
Marwa Zafarullah ◽  
Mittal Jasoliya ◽  
Flora Tassone
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Fragile X Syndrome ◽  
Cell Lines ◽  
Behavioral Problems ◽  
Cell Model ◽  
Fragile X ◽  
Healthy Controls ◽  
Full Mutation ◽  
Epithelial Cell Lines

Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition associated with intellectual disability and behavioral problems due to the lack of the Fragile X mental retardation protein (FMRP), which plays a crucial role in synaptic plasticity and memory. A desirable in vitro cell model to study FXS would be one that can be generated by simple isolation and culture method from a collection of a non-invasive donor specimen. Currently, the various donor-specific cells can be isolated mainly from peripheral blood and skin biopsy. However, they are somewhat invasive methods for establishing cell lines from the primary subject material. In this study, we characterized a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). The urine-derived cells expressed epithelial cell surface markers via fluorescence-activated cell sorting (FACS). We observed inter, and the intra-tissue CGG mosaicism in the PBMCs and the urine-derived cells from participants with FXS potentially related to the observed variations in the phenotypic and clinical presentation FXS. We characterized these urine-derived epithelial cells for FMR1 mRNA and FMRP expression and observed some expression in the lines derived from full mutation mosaic participants. Further, FMRP expression was localized in the cytoplasm of the urine-derived epithelial cells of healthy controls. Deficient FMRP expression was also observed in mosaic males, while, as expected, no expression was observed in cells derived from participants with a hypermethylated full mutation.

330 REDUCED HYPERACUTE REJECTION BY TRIPLE TRANSGENIC EXPRESSION OF HUMAN COMPLEMENT REGULATORY FACTORS (hDAF and hCD59) AND H-TRANSFERASE

2011 ◽  
Vol 23 (1) ◽  
pp. 261
Author(s):  
Y. H. Jeong ◽  
G. H. Jang ◽  
I. S. Hwang ◽  
C. H. Park ◽  
H. J. Lee ◽  
...  
Keyword(s):  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Vector ◽  
Inhibitory Effect ◽  
Hyperacute Rejection ◽  
Stable Cell Line ◽  
Control Group ◽  
Human Complement ◽  
Transgenic Cell

The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.

scholarly journals FAN1 nuclease processes and pauses on disease-associated slipped-DNA repeats: Mechanism against repeat expansions

2021 ◽  
Author(s):  
Amit Laxmikant Deshmukh ◽  
Marie-Christine Caron ◽  
Mohiuddin Mohiuddin ◽  
Stella Lanni ◽  
Gagan B. Panigrahi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Disease Onset ◽  
Repeat Expansion ◽  
Dna Repeats ◽  
The Third ◽  
Dna Repair Proteins ◽  
Repeat Expansions ◽  
Specific Ligand

FAN1 nuclease is a modifier of repeat expansion diseases, including Huntington's disease (HD), fragile X syndrome, and autism. The age of HD onset correlates with ongoing 'inchworm-like' repeat expansions (1-3 CAG units/event) in HD brains, and is regulated by three modifiers: The first two, repeat tract length and purity exert their effects by enhancing and slowing CAG expansions, respectively, by affecting the formation of slipped-DNAs - mutagenic intermediates of instability; which are processed to expansions by the third modifiers, DNA repair proteins. FAN1 protects against hyper-expansions of repeats, by unknown mechanisms. We show FAN1, through iterative cycles bound, dimerized and cleaved slipped-DNAs, yielding striking patterns of distinct exo-nuclease pauses along slip-outs; 5′-C↓A↓GC↓A↓G-3′ and 5′-C↓T↓G↓C↓T↓G-3′. The transcriptionally-displaced CAG strand was excised slower than its complementary CTG strand, required A·A and T·T mismatches, as fully-paired hairpins arrested excision progression, while disease-delaying CAA interruptions further slowed FAN1 excision. In contrast, endo-nucleolytic cleavage was insensitive to slip-outs. Rare FAN1 variants were found in autism individuals with CGG/CCG repeat expansions. Excision of CGG/CCG slip-outs were similarly excised, with CGG being slower than CCG. The slip-out specific ligand, Naphthyridine-Azaquinolone, shown to induce contractions of expanded repeats in cells, required FAN1 for its effect, and protected slip-outs from FAN1's exo- but not endo-nucleolytic digestion. FAN1's 'inchworm' pausing of slip-out excision is suited to minimize incremental expansions and modulating disease onset.

scholarly journals Genetic Transformation of LoHDZ2 and Analysis of its Function to Enhance Stress Resistance in Larch

2021 ◽  
Author(s):  
Peiqi An ◽  
Ruofan Qin ◽  
Qingrong Zhao ◽  
Xuefeng Li ◽  
Chen Wang ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Differentially Expressed Genes ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type Cell ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gus Staining ◽  
Transgenic Cell

Abstract To study the function of LoHDZ2 in larch, we first constructed a p1302-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured.The results are summarized below.1. When the specific fragment of the target gene in the genome of the resistant cell line was amplified, at the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines.2. To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 downregulated ones) were subjected to qRT–PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-seq, indicating that the transcriptome data were reliable.3. The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were downregulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process.4. After PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced.In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2, which is a member of the HD-ZipII subfamily, of Larix olgensis may participate in the response of plants to the external environment and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.

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