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Animal Models - GEG Tech top picks
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BigField GEG Tech's insight:
The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. 
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Mosaic mutations and off-target effects caused by CRISPR/Cas9 have led to concerns about the efficiency and specificity of this new technique in non-human primates and other large animals. Here the authors discuss recent findings from primate embryos, with a focus on the technical issues CRISPR/Cas9 faces before producing non-human primate models of human diseases.
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BigField GEG Tech's insight:
In this study, the scientists show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA.
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In this study, the scientists use CRISPR system to generate mice carrying the inactivation (via deletion) of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus.These results illustrate how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be validated.
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In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited significantly prolonged survival further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds.
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In this study, the scientists produced human lactoferrin (hLF) and human lysozyme (hLZ) bi-transgenic pigs and assessed the milk’s antibacterial ability. Both the hLF and hLZ were expressed in the mammary gland of bi-transgenic pigs, as detected by western blotting. The expression amounts were 6.5 g/L for hLF and 1.1 mg/L for hLZ (ELISA).
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In transgenesis manual injection of the components into the pronuclei or the cytoplasm of zygotes could be a bottleneck. To overcome it, the scientists employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 mRNA, sgRNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted NHEJ and HDR mutations with high efficiency.
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The authors studied effects of liraglutide in adolescent pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) in the beta-cells, leading to a pre-diabetic condition including disturbed glucose tolerance, reduced insulin secretion and progressive reduction of functional beta-cell mass.
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The scientists constructed a mini-pig model by mono-allelic knockout (KO) of PKD1 using zinc finger nuclease. After approximately six months, renal cysts appeared and grew progressively in the KO pigs. Contrast-enhanced computed tomography confirmed that all of the mutant pigs had renal and hepatic cysts, when they were 11-month-old. Therefore, the PKD1 mono-allelic knockout is sufficient to trigger renal cystogenesis, and this pig model may provide a platform for future study of renal cyst formation.
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The scientists report skeletal defects in Osterix-Cre (Osx-Cre) transgenic mice including delayed calvarial ossification and fracture calluses at multiple skeletal sites. These data suggest that Osx-Cre containing controls should be used for both in vivo and in vitro skeletal analyses of conditional knockout mice generated with this Osx-Cre mouse strain.
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The authors describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. They confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.
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The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
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The authors reported an ultrastructural study of ksr2 −/− mice spermatogenesis. Remarkable findings regard the altered spermiogenetic process concomitant with a severe disorganization of interstitial tissue. Further studies are needed to assess the ksr2 −/− mice hormonal status, focussing on testosterone levels since the interstitial tissue, where the Leydig cells reside, was compromised.
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In this study, the scientists constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. From 669 injected eggs, 218 chickens were successfully hatched. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml.
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BigField GEG Tech's insight:
Injection of recombinant Cas9 protein and synthetic guide RNAs into mouse zygotes has been shown to facilitate gene disruption and knock-ins using the CRISPR system. These technologies may soon displace genetic modification using embryonic stem cells.
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BigField GEG Tech's insight:
The authors developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems. They demonstrated that injection of up to three different cassettes in a single injection session allows to obtain multiple transgenic mice with a very high efficency (up to 62%).
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The scientists used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle.
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BigField GEG Tech's insight:
The authors report the efficient creation of an Apolipoprotein E knockout rabbit by using zinc finger nucleases. The knockout rabbits had drastically elevated cholesterol and moderately increased triglyceride levels, mimicking symptoms in human heart disease.
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BigField GEG Tech's insight:
Liao et al. transfected human cell lines with Cas9 and with gRNAs targeting the long terminal repeats (LTRs) of HIV-1, which led to reduced infection of these cells by HIV-1, compared with wild-type cells. Similarly, CRISPR–Cas9 transfection into cell lines with integrated virus reduced the levels of proviral DNA. Furthermore, human T cell lines engineered to stably express Cas9 and LTR-specific gRNAs maintained a low level of viral infection even 14 days post-infection with HIV-1. Finally, the authors showed that CRISPR–Cas9 efficiently reduced virus production in primary human cells, demonstrating that expression of the CRISPR–Cas9 system in humans confers protection against HIV-1 infection.
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The scientists describe viable gene-targeted pigs carrying a latent KRAS G12D mutant allele that can be activated by Cre recombination.
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BigField GEG Tech's insight:
Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient’s immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.
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This study established an improved model system for Acute myeloid leukemia that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes.
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The authors injected TALEN-mRNAs into the zygotes with pronucleus micromanipulation technique, and obtained seven Foxo3 mutants of 20 mice (35%). All seven were heterozygous mutants and the gene mutagenesis rates of the mice. The comportemental and histological examinations suggested that the mutation of Foxo3 severely affected the fertile ability of female and perhaps male in some degree.
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The authors describe viable gene-targeted pigs carrying a latent KRAS G12D mutant allele that can be activated by Cre recombination. Cre-activated KRAS G12D animals add to a growing set of gene-targeted pigs that includes a Cre-activated oncogenic mutant TP53, a Cre-responsive dual fluorescent reporter and two truncating mutations of APC. These alleles can be combined and activated in various tissues to produce new models for cancer research. www.geg-tech.com
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In this review the authors described how CRISPR-Cas9 has been used to create germline and somatic mouse models with point mutations, deletions and complex chromosomal rearrangements. They highlight the progress and challenges of such approaches, and how these models can be used to understand the evolution and progression of individual tumors and identify new strategies for cancer treatment.
www.geg-tech.com/Vectors