Animal Models - GEG Tech top picks
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Could Stem Cells eventually replace Animal Testing in Drug Development?

Could Stem Cells eventually replace Animal Testing in Drug Development? | Animal Models - GEG Tech top picks | Scoop.it
The European Biotech News Website
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Animal testing is a crucial part of developing new medicines, but it is accompanied by a host of ethical concerns. Stem cells could be one of the alternatives, according to the European Commission’s Joint Research Center on alternative methods to animal experimentation in research and regulation. While attention to stem cells has been focused on potential in regenerative medicine, they have also earned a reputation in R&D for their capacity to produce human tissues and to model diseases. Indeed, they could lead to the development of more ethical, efficient and economical tools that could fast track R&D.

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Generation and analysis of the Rett syndrome-associated MeCP2- null rat model

Generation and analysis of the Rett syndrome-associated MeCP2- null rat model | Animal Models - GEG Tech top picks | Scoop.it

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In order to investigate the role of MeCP2 in brain development and RTT pathogenesis, we aimed to set up the MeCP2-null rat model using the CRISPR/Cas9 technology. Their results indicated that the MeCP2 knockout rats showed body weight loss, anxiety tendency and cognitive deficits. The MeCP2-null rat model established in this study recapitulates the major symptoms of RTT patients and provides an alternative tool for future studies of MeCP2 functions.

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Biallelic β-carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9 

Biallelic β-carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9  | Animal Models - GEG Tech top picks | Scoop.it
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Here, the authors show that biallelic modification of BCO2 resulted in yellow fat, compared with the fat color in monoallelic individuals and wild types (snow-flower white). These results indicate that genetic modification via CRISPR/Cas9 holds great potential for validating gene functions as well as for generating desirable phenotypes for economically important traits in livestock.

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Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer

Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer | Animal Models - GEG Tech top picks | Scoop.it
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Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids designed to target exon 6 of porcine GGTA1.

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Phenotypic characterization of a novel HO-1 depletion model in the rat

Phenotypic characterization of a novel HO-1 depletion model in the rat | Animal Models - GEG Tech top picks | Scoop.it
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The present study describes the generation of a HO-1 deficient Hmox1−/− rat model and characterizes its renal and extrarenal phenotype. Hmox1−/− rats had growth retardation and splenomegaly compared to their Hmox1+/+ littermates. Focal segmental glomerulosclerosis-type lesions and interstitial inflammatory infiltrates were prominent morphologic findings and were associated with increased blood urea nitrogen, serum creatinine and albuminuria. There was no increase in iron deposition in glomeruli, tubules or interstitium. Iron deposition in spleen and liver was reduced. Electron microscopic examination of glomeruli revealed edematous podocytes with scant areas of foot process effacement but otherwise well preserved processes and slit-diaphragms. Of the filtration barrier proteins examined, β-catenin expression was markedly reduced both in glomeruli and extrarenal tissues. Since the rat is the preferred laboratory animal in experimental physiology and pathophysiology, the rat model of HO-1 deficiency may provide a novel tool for investigation of the role of this enzyme in renal function and disease.

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A new transgenic mouse model for conditional overexpression of the Polycomb Group protein EZH2

A new transgenic mouse model for conditional overexpression of the Polycomb Group protein EZH2 | Animal Models - GEG Tech top picks | Scoop.it
The Polycomb Group protein EZH2 is upregulated in most prostate cancers, and its overexpression is associated with poor prognosis. Most insights into the functional role of EZH2 in prostate cancer hav
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In this study, the authors  developed one such transgenic mouse model for conditional overexpression of Ezh2. In this transgene, Ezh2 and Luciferase are transcribed from a single open reading frame. They report the generation of a wildtype Ezh2 overexpression mouse model that allows for intravital surveillance of tissues with activated transgene. This model will be an invaluable tool for further unravelling the role of EZH2 in cancer.

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Rapamycin Use Increased Lifespan in Leigh Syndrome Animal Models

Rapamycin Use Increased Lifespan in Leigh Syndrome Animal Models | Animal Models - GEG Tech top picks | Scoop.it
Results of animal studies suggest that rapamycin may be a therapeutic strategy for Leigh syndrome, and possibly other mitochondria diseases.
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Both mouse and fruit fly animal models of Leigh syndrome suggest that blocking a specific pathway controlled by the protein TOR increases animals’ lifespan. The results suggest that a future therapeutic strategy for Leigh syndrome, and possibly other mitochondria diseases, may require TOR inhibition.

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Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

Disruption of  FGF5  in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers | Animal Models - GEG Tech top picks | Scoop.it
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By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced.

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Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs | Animal Models - GEG Tech top picks | Scoop.it
The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing res
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In this study, the scientists designed pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 which were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7. Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92–100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls. There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.

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SA Scientists Developing Animal Models For Zika Testing

SA Scientists Developing Animal Models For Zika Testing | Animal Models - GEG Tech top picks | Scoop.it
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San Antonio scientists are part of a push to develop laboratory animal models to study the Zika virus. Baboons and monkeys may be key to unlocking new treatments and vaccines.

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Exome sequencing in the knockin mice generated using the CRISPR/Cas system

Exome sequencing in the knockin mice generated using the CRISPR/Cas system | Animal Models - GEG Tech top picks | Scoop.it
Knockin (KI) mouse carrying a point mutation has been an invaluable tool for disease modeling and analysis.
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In this study, the authors performed whole exome sequencing of multiple lines of F1 heterozygous Ntrk1 KI mice generated using the CRISPR/Cas system in comparison to that of a wild-type mouse used as a control. We found three nucleotide insertions and/or deletions (InDels) in four KI mice but not in a control mouse. In vitro digestion assay suggested that each InDel occurred as a de novo mutation, was carried-over from the parental mice, or was incorporated through the Cas9 nuclease mediated off-target cleavage. These results suggest that frequency of InDels found in KI mice generated by the CRISPR/Cas technology is not high, but cannot be neglected and careful assessment of these mutations is warranted.

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Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System

Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System | Animal Models - GEG Tech top picks | Scoop.it
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The results of this study showed that CRISPR/Cas9 system could be employed to target the same sites in different species, when sgRNAs are designed within conserved regions, and therefore will be critically important and applicable for human disease model.

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Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation

Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation | Animal Models - GEG Tech top picks | Scoop.it
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The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified pigs were created through CRISPR/Cas9-directed mutation and human transgene delivery. They find that silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.

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A mouse model for MERS coronavirus-induced acute respiratory distress syndrome

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome | Animal Models - GEG Tech top picks | Scoop.it
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Mice made susceptible to MERS-CoV, using CRISPR–Cas9 to alter the gene encoding the dipeptidyl peptidase 4 receptor, allow efficient viral replication in the lungs and display symptoms indicative of severe acute respiratory stress.

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An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome

An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome | Animal Models - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

In this wotk, the authors used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes their analysis of the genomes of these enhancer knockout lines to detect possible off-target effects.

They conclued that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome.

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TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna

TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna | Animal Models - GEG Tech top picks | Scoop.it
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Here, the scientists confirmed TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna, a model organism for ecological and toxicological genomics. They obtained H2B-GFP expression in Daphnia with a germline transmission efficiency of 3%. Of the three transgenic animals generated, two had donor DNA at the targeted genomic site, which suggested concurrent cleavage of the injected plasmid DNA and genome DNA. The availability of such tools that are capable of targeted knock-in of foreign genes will be extremely useful for advancing the knowledge of gene function and contribute to an increased understanding of functional genomics in Daphnia.

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Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors

Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors | Animal Models - GEG Tech top picks | Scoop.it

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BigField GEG Tech's insight:

This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively.  The results show that mutant pigs could be generated by direct pronuclear microinjection of CRISPR vectors and that the 4-site strategy has a better mutant efficiency.

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The transgenic expression of human follistatin-344 increases skeletal muscle mass in pigs

The transgenic expression of human follistatin-344 increases skeletal muscle mass in pigs | Animal Models - GEG Tech top picks | Scoop.it
Follistatin (FST), which was first found in the follicles of cattle and pigs, has been shown to be an essential regulator for muscle development. Mice that were genetically engineered to overexpress F
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Here, the scientists describe transgenic Duroc pigs that exogenously express Fst specifically in muscle tissue. Their findings indicate that muscle-specific Fst overexpression in pigs enhances skeletal muscle growth, at least partly due to myofiber hypertrophy and providing a promising approach to increase muscle mass in pigs and other livestock.

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New Animal Model of Epilepsy Could Help Develop Novel AEDs | Epilepsy Research UK

New Animal Model of Epilepsy Could Help Develop Novel AEDs | Epilepsy Research UK | Animal Models - GEG Tech top picks | Scoop.it
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Researchers at Florida Atlantic University and The Scripps Research Institute,  in the US, have developed a new animal model of epilepsy that will allow the screening of hundreds of thousands of potential antiepileptic compounds. This work is published in the leading scientific journal, Plos One

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How gene editing is changing what a lab animal looks like

How gene editing is changing what a lab animal looks like | Animal Models - GEG Tech top picks | Scoop.it
What makes a good animal model? New techniques bring opportunities and challenges to model organisms.
BigField GEG Tech's insight:

Different types of studies use different stand-ins: Flies for genetics; zebrafish for early development; rats and mice and monkeys for cancer, neuroscience and more.

Now, new techniques such as gene editing mean that scientists can probe and alter the genes of any animal. The methods open the door for new organisms — such as squid and octopuses — to join scientists’ basic toolkits. With these new arrivals come new questions. What is needed for a good animal model, and how are gene-snipping tools changing the game?

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Pronuclear Injection-Based Targeted Transgenesis 

Pronuclear Injection-Based Targeted Transgenesis  | Animal Models - GEG Tech top picks | Scoop.it
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Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models but is limited by the random integration of the DNA of interest into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression.

A new method, called Pronuclear Injection-based Targeted Transgenesis (PITT), employs an enzymatic transfer of exogenous DNA from a donor vector to a previously created landing-pad site in the mouse genome. DNA transfer is achieved using molecular tools such as the Cre-LoxP recombinase and PhiC31-attB/P integrase systems. Here, the authors provide protocols for performing PITT and an overview of the current PITT tools available to the research community.

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Effects of Melanocortin 3 and 4 Receptor Deficiency on Energy Homeostasis in Rats

Effects of Melanocortin 3 and 4 Receptor Deficiency on Energy Homeostasis in Rats | Animal Models - GEG Tech top picks | Scoop.it
Melanocortin-3 and 4 receptors (MC3R and MC4R) can regulate energy homeostasis, but their respective roles especially the functions of MC3R need more exploration.
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Here Mc3r and Mc4r single and double knockout (DKO) rats were generated using CRISPR-Cas9 system. Their data demonstrated MC3R deficiency caused a reduction of food intake and body weight, whereas at the same time exhibited additive effects on top of MC4R deficiency on lipid and glucose metabolism. This is the first phenotypic analysis and systematic comparison of Mc3r KO, Mc4r KO and DKO rats on a homogenous genetic background. These mutant rats will be important in defining the complicated signalling pathways of MC3R and MC4R. Both Mc4r KO and DKO are good models for obesity and diabetes research.

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Progressive muscle proteome changes in a clinically relevant pig model of Duchenne muscular dystrophy

Progressive muscle proteome changes in a clinically relevant pig model of Duchenne muscular dystrophy | Animal Models - GEG Tech top picks | Scoop.it
Duchenne muscular dystrophy (DMD) is caused by genetic deficiency of dystrophin and characterized by massive structural and functional changes of skeletal muscle tissue, leading to terminal muscle failure.
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Scientists recently generated a novel genetically engineered pig model reflecting pathological hallmarks of human DMD better than the widely used mdx mouse. To get insight into the hierarchy of molecular derangements during DMD progression, they performed a proteome analysis of biceps femoris muscle samples from 2-day-old and 3-month-old DMD and wild-type (WT) pigs. The extent of proteome changes in DMD vs. WT muscle increased markedly with age, reflecting progression of the pathological changes. In 3-month-old DMD muscle, proteins related to muscle repair such as vimentin, nestin, desmin and tenascin C were found to be increased, whereas a large number of respiratory chain proteins were decreased in abundance in DMD muscle, indicating serious disturbances in aerobic energy production and a reduction of functional muscle tissue. The combination of proteome data for fiber type specific myosin heavy chain proteins and immunohistochemistry showed preferential degeneration of fast-twitch fiber types in DMD muscle. The stage-specific proteome changes detected in this large animal model of clinically severe muscular dystrophy provide novel molecular readouts for future treatment trials.

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CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities

CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities | Animal Models - GEG Tech top picks | Scoop.it
Chickens are an invaluable model for studying human diseases, physiology and especially development, but have lagged in genetic applications.
BigField GEG Tech's insight:

In this study, the authors show that the CRISPR/Cas9 system can precisely edit the chicken genome. They generated HIRA, TYRP1, DICER, MBD3, EZH2, and 6 other gene knockouts in two chicken cell lines using the CRISPR/Cas9 system, with no off-target effects detected. They also showed that very large deletions (>75 kb) and modification by homology-directed repair could be achieved.

These findings demonstrate that precisely targeted genetic manipulation of the genome using the CRISPR/Cas9 system can be extended to the highly adaptable in vivo chicken embryo model.

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Somatic cell reprogramming-free generation of genetically modified pigs

Somatic cell reprogramming-free generation of genetically modified pigs | Animal Models - GEG Tech top picks | Scoop.it
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Here, scientists provide a simple method for CRISPR/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

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