scholarly journals Mouse Models of Achromatopsia in Addressing Temporal “Point of No Return” in Gene-Therapy

2021 ◽  
Vol 22 (15) ◽  
pp. 8069
Author(s):  
Nan-Kai Wang ◽  
Pei-Kang Liu ◽  
Yang Kong ◽  
Sarah R. Levi ◽  
Wan-Chun Huang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mouse Models ◽  
Normal Cone ◽  
Es Cells ◽  
Embryonic Stem ◽  
Therapeutic Window ◽  
Cone Response ◽  
Cone Function ◽  
Point Of No Return ◽  
Stop Cassette

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form “functional” CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

scholarly journals The Mutant Mouse Resource and Research Center (MMRRC): the NIH-supported National Public Repository and Distribution Archive of Mutant Mouse Models in the USA

2021 ◽  
Author(s):  
James Amos-Landgraf ◽  
Craig Franklin ◽  
Virginia Godfrey ◽  
Franziska Grieder ◽  
Kristin Grimsrud ◽  
...  
Keyword(s):  
Mouse Models ◽  
Disease Surveillance ◽  
Mutant Mouse ◽  
Disease Modeling ◽  
Es Cells ◽  
Genetic Research ◽  
Embryonic Stem ◽  
Research Center ◽  
The Usa ◽  
Mouse Lines

AbstractThe Mutant Mouse Resource and Research Center (MMRRC) Program is the pre-eminent public national mutant mouse repository and distribution archive in the USA, serving as a national resource of mutant mice available to the global scientific community for biomedical research. Established more than two decades ago with grants from the National Institutes of Health (NIH), the MMRRC Program supports a Consortium of regionally distributed and dedicated vivaria, laboratories, and offices (Centers) and an Informatics Coordination and Service Center (ICSC) at three academic teaching and research universities and one non-profit genetic research institution. The MMRRC Program accepts the submission of unique, scientifically rigorous, and experimentally valuable genetically altered and other mouse models donated by academic and commercial scientists and organizations for deposition, maintenance, preservation, and dissemination to scientists upon request. The four Centers maintain an archive of nearly 60,000 mutant alleles as live mice, frozen germplasm, and/or embryonic stem (ES) cells. Since its inception, the Centers have fulfilled 13,184 orders for mutant mouse models from 9591 scientists at 6626 institutions around the globe. Centers also provide numerous services that facilitate using mutant mouse models obtained from the MMRRC, including genetic assays, microbiome analysis, analytical phenotyping and pathology, cryorecovery, mouse husbandry, infectious disease surveillance and diagnosis, and disease modeling. The ICSC coordinates activities between the Centers, manages the website (mmrrc.org) and online catalog, and conducts communication, outreach, and education to the research community. Centers preserve, secure, and protect mutant mouse lines in perpetuity, promote rigor and reproducibility in scientific experiments using mice, provide experiential training and consultation in the responsible use of mice in research, and pursue cutting edge technologies to advance biomedical studies using mice to improve human health. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest standards of rigor and reproducibility, while donating investigators benefit by having their mouse lines preserved, protected, and distributed in compliance with NIH policies.

Lentiviral Insertion Site Analysis of Corrected Mouse Model of β Thalassemia Following Lentiviral Transduction of Embryonic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2592-2592
Author(s):  
Rui Yang ◽  
Sean C. McConnell ◽  
Yongliang Huo ◽  
Clayton Ulrey ◽  
Shan-Run Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Lentiviral Transduction ◽  
Insertion Sites ◽  
Real Time Qpcr

Abstract The delivery of therapeutic transgenes via lentiviral transduction of stem cells holds great promise for future cell based therapies for inherited genetic disorders. The regulation of any transgene integrated into patient derived embryonic stem (ES) cells (generated via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. Because genetically manipulated ES cells can be clonally isolated, expanded to great numbers in an undifferentiated state, and differentiated to specific cell types, they can be carefully tested in vitro prior to their reintroduction back into patients. ES cells derived from a mouse model of β thalassemia were transduced with a recombinant, self inactivating (SIN) lentiviral vector containing a 2.3 kb human β-globin gene and a 3.2 kb Locus Control Region composed of regulatory elements; HS2, HS3, and HS4. Clonal populations of transfected β thalassemic ES cells were isolated, archived, and utilized to produce genetically identical offspring by injection into tetraploid blastocysts or eight-cell embryos. Real-Time QPCR expression analyses demonstrated high levels of human β-globin gene expression in the peripheral blood of five of the six lines produced. The average expression level per transgene copy number ranged from 13% to 62% of endogenous mouse α-globin levels. Peripheral blood hemolysates analyzed by HPLC confirmed the high level production of human β-globin chains. Measurement of the red blood indices in these five lines showed that the anemia was corrected. In order to examine the influence of chromosomal position effects on individual transgene expression, the lentiviral insertion sites were mapped by linear amplification (LAM) PCR. Cloned mice from the six lines were bred in order to segregate each of their individual lentiviral transgenes. Offspring containing single insertion sites were analyzed for human β-globin transgene expression and synthesis by Real-Time QPCR of blood RNA and by HPLC of hemolysates, respectively. These analyses demonstrate that there is a preference for provirus integration in ES cells; all transgene insertions are not expressed equally; lentiviral transduction of a human β-globin gene into ES cells can cure a severe hemoglobinopathy; and that the delivery of therapeutic transgenes via lentiviral transduction of ES cells did not harm the developmental potential of the cells to produce an entire mouse.

scholarly journals Efficient Generation of Mice with Consistent Transgene Expression by FEEST

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Lei Gao ◽  
Yonghua Jiang ◽  
Libing Mu ◽  
Yanbin Liu ◽  
Fengchao Wang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Mouse Models ◽  
Transgene Expression ◽  
Es Cells ◽  
Mouse Line ◽  
Transgenic Mouse Models ◽  
Transgenic Mouse Line ◽  
A Genome

Abstract Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.

A Novel Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia Locates the Erythroid Defect at CFU-E / Proerythroblast Transition.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 178-178 ◽  
Author(s):  
Pekka Jaako ◽  
Johan Flygare ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mouse Models ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Proliferative Potential ◽  
Down Regulation ◽  
Diamond Blackfan Anemia ◽  
Doxycycline Treatment ◽  
Forming Defects

Abstract Abstract 178 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Presently, many different DBA disease genes are known that all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). In order to study pathophysiology and to evaluate novel therapies, DBA animal models are needed. Since RNA interference -mediated RPS19 down regulation has been shown to result in a DBA phenotype in human cells in vitro, we decided to use the short hairpin RNA (shRNA) technology to create an RPS19-deficient mouse model for DBA. We designed miR30 -styled shRNAs against RPS19 and introduced them into mouse embryonic stem (ES) cells downstream of the collagen A1 locus using site-specific recombination. The resulting ES cell clones contain a single RPS19-targeting shRNA under the control of a doxycycline-responsive promoter. We have generated and characterized two mouse models expressing different RPS19-targeting shRNAs (shRNA-B and shRNA-D). In general, this system allows an inducible and dose-dependent regulation of shRNA expression providing an ideal tool to study conditions like DBA that are caused by haploinsufficient expression of a protein. To induce the expression of RPS19-targeting shRNAs mice were fed with doxycycline administered in drinking water. Induction of the shRNA-B construct had no effect on the erythrocyte level, hemoglobin concentration or hematocrit, although we saw a gradual elevation in mean corpuscular volume (MCV) and a decrease in platelet number. However, after 25–35 days of doxycycline treatment the mice homozygous for the shRNA-B underwent severe weight loss accompanied with a reduction in erythrocyte number and ultimately died. In contrast, shRNA-D mice exhibited decreased erythrocyte number, hemoglobin and hematocrit already after a 10 day doxycycline treatment. When the induction was kept on longer, the homozygous mice developed a more severe anemia and died around day 20, while the heterozygous mice were able to compensate the blood indices back to normal level. In spite of the differences in blood phenotypes, both models had a similar FACS phenotype revealing a profound decrease in the number of proerythroblasts, while the levels of erythroid and bipotential megakaryocytic-erythroid (MegE) progenitors were normal or increased. We also saw an accumulation of the late erythroid precursors. Proliferative potential of erythroid progenitors was evaluated at a clonal level in vitro. When MegE or CFU-E progenitors from induced mice were cultured in presence of doxycycline, the number and size of the erythroid clones were decreased compared to controls. However, when RPS19 expression was restored by culturing the cells without doxycycline, the observed proliferation defect of MegE clones was completely restored while the rescue of CFU-E clones was only partial. We also noticed that RPS19-deficient megakaryocytes appeared smaller in size compared to controls. Importantly, transduction of RPS19-deficient cells with a lentiviral vector overexpressing sequence-modified RPS19 cDNA rescued the proliferation and colony-forming defects in vitro demonstrating that the erythroid phenotype is specifically due to down regulation of RPS19. In summary, we have generated two novel mouse models for RPS19-deficient DBA that recapitulate the key erythroid phenotype seen in patients based on both FACS analysis and single-cell proliferation assays. These models will serve as a good tool to determine the molecular mechanisms responsible for DBA and also to test gene replacement therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Haplolethal Locus Uncovered by Deletions in the Mouse t Complex

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 675-682
Author(s):  
Victoria L Browning ◽  
Rebecca A Bergstrom ◽  
Sandra Daigle ◽  
John C Schimenti
Keyword(s):  
Gene Dosage ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromosome 17 ◽  
Mammalian Development ◽  
T Complex ◽  
Segmental Aneuploidy ◽  
Systematic Exploration ◽  
Radiation Induced ◽  
Altered Gene

Abstract Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.

scholarly journals Intranasal oxytocin administration ameliorates social behavioral deficits in a POGZWT/Q1038R mouse model of autism spectrum disorder

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kohei Kitagawa ◽  
Kensuke Matsumura ◽  
Masayuki Baba ◽  
Momoka Kondo ◽  
Tomoya Takemoto ◽  
...  
Keyword(s):  
Autism Spectrum Disorder ◽  
Social Behavior ◽  
Mouse Model ◽  
Mouse Models ◽  
De Novo ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
De Novo Mutation ◽  
Behavioral Deficits

AbstractAutism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by core symptoms of impaired social behavior and communication. Recent studies have suggested that the oxytocin system, which regulates social behavior in mammals, is potentially involved in ASD. Mouse models of ASD provide a useful system for understanding the associations between an impaired oxytocin system and social behavior deficits. However, limited studies have shown the involvement of the oxytocin system in the behavioral phenotypes in mouse models of ASD. We have previously demonstrated that a mouse model that carries the ASD patient-derived de novo mutation in the pogo transposable element derived with zinc finger domain (POGZWT/Q1038R mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZWT/Q1038R mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZWT/Q1038R mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZWT/Q1038R mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZWT/Q1038R mice and that of WT mice. A chromatin immunoprecipitation assay revealed that POGZ binds to the promoter region of OXTR and is involved in the transcriptional regulation of OXTR. In summary, our study demonstrate that the pathogenic mutation in the POGZ, a high-confidence ASD gene, impairs the oxytocin system and social behavior in mice, providing insights into the development of oxytocin-based therapeutics for ASD.

scholarly journals The Function of Selenium in Central Nervous System: Lessons from MsrB1 Knockout Mouse Models

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1372
Author(s):  
Tengrui Shi ◽  
Jianxi Song ◽  
Guanying You ◽  
Yujie Yang ◽  
Qiong Liu ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Synaptic Plasticity ◽  
Mouse Model ◽  
Mouse Models ◽  
Knockout Mouse ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Knockout Mouse Model ◽  
The Central Nervous System

MsrB1 used to be named selenoprotein R, for it was first identified as a selenocysteine containing protein by searching for the selenocysteine insert sequence (SECIS) in the human genome. Later, it was found that MsrB1 is homologous to PilB in Neisseria gonorrhoeae, which is a methionine sulfoxide reductase (Msr), specifically reducing L-methionine sulfoxide (L-Met-O) in proteins. In humans and mice, four members constitute the Msr family, which are MsrA, MsrB1, MsrB2, and MsrB3. MsrA can reduce free or protein-containing L-Met-O (S), whereas MsrBs can only function on the L-Met-O (R) epimer in proteins. Though there are isomerases existent that could transfer L-Met-O (S) to L-Met-O (R) and vice-versa, the loss of Msr individually results in different phenotypes in mice models. These observations indicate that the function of one Msr cannot be totally complemented by another. Among the mammalian Msrs, MsrB1 is the only selenocysteine-containing protein, and we recently found that loss of MsrB1 perturbs the synaptic plasticity in mice, along with the astrogliosis in their brains. In this review, we summarized the effects resulting from Msr deficiency and the bioactivity of selenium in the central nervous system, especially those that we learned from the MsrB1 knockout mouse model. We hope it will be helpful in better understanding how the trace element selenium participates in the reduction of L-Met-O and becomes involved in neurobiology.

scholarly journals The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

2021 ◽  
Vol 7 (4) ◽  
pp. eabb9149
Author(s):  
Zhijun Huang ◽  
Jiyoung Yu ◽  
Wei Cui ◽  
Benjamin K. Johnson ◽  
Kyunggon Kim ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Dna Demethylation ◽  
Chromosomal Protein ◽  
Dna Hypomethylation ◽  
Tet Proteins ◽  
Target Sites ◽  
Structural Maintenance Of Chromosomes ◽  
Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

scholarly journals Retinoic acid signaling is critical during the totipotency window in early mammalian development

2021 ◽  
Author(s):  
Ane Iturbide ◽  
Mayra L. Ruiz Tejeda Segura ◽  
Camille Noll ◽  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Networks ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mammalian Development ◽  
Cell Stage ◽  
Cell Potential ◽  
Cellular Models ◽  
Early Embryos ◽  
Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

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