Animal Models - GEG Tech top picks

Here the scientists used CRISPR system to generate the first mutant lines in ants by disrupting orco, a gene required for the function of all ORs. They find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting that they are unable to perceive pheromones.

In this work, the scientists propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, they showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

In this study, the Sprague Dawley rat iPSCs were generated using lentiviral method. The p53 gene was targeted in rat iPSCs using homologous recombination mediated by P53 zinc-finger nucleases (ZFNs). The results of this work showed that these rat iPSCs were pluripotent status. P53 gene was targeted successfully with high efficiency by coelectroporating the donor targeting vectors and p53 ZFN vector to these rat iPSCs.

The $38 million Series A round announced by Cambridge, MA-based firm eGenesis comes as the field of human-compatible organs grown in animals, while still years from real products, is back in play because of advances in new biological modification techniques including the genome editing tool CRISPR-Cas9.

Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects.

Increasing cashmere yield is one of the vital aims of cashmere goats breeding.

For the present study, donor fibroblasts were stably transfected via a piggyBac (PB) donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). These results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.

Rats were the first mammalian species domesticated for scientific purposes, and they soon became the most widely used animal model in biomedical sciences, including cardiovascular research and behavioral neuroscience. Yet, after the development of technologies to manipulate genes, researchers largely shifted to the use of mice. However, as the authors lay out with examples from drug addiction, social behavior, and cardiovascular research, rats have experimental advantages over mice. With the introduction of zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) methodologies, and, specifically, the clustered regularly interspaced short palindromic repeats (CRISPR) associated system, gene targeting is no longer limited to mice. Development of genetic technologies in rats allows researchers to take advantage of the unique opportunities offered by this species in biomedical research.

David Ishee’s plan was simple, if not exactly free of complication. From the shed that functions as his laboratory in rural Mississippi, he hoped to use genetic engineering to rid dogs of the types of terrible disorders caused by decades of high-end breeding.

Now, using a novel PET radiotracer called Neuroflux, a team of researchers from the Athinoula A. Martinos Center for Biomedical Imaging at Massachusetts General Hospital (MGH) and the MassGeneral Institute of Neurodegenerative Disease has found a way to quantify olfactory sensory neurons and thus improve measurements of olfactory health. They describe their findings in the February issue of The Journal of Clinical Investigation.

Studies in animal models have proven extremely useful in understanding the mechanisms of many human diseases and in assessing new therapeutic strategies.

But current animal models of human neurological diseases associated with aging, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), may not recapitulate key aspects of disease pathology. Recently, investigators have found that dog models of AD may present a truer, more translatable picture of the disease than genetic mouse models.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. In this work, the authors transduced tree shrew spermatogonial stem cells (SSCs) with (EGFP)-expressing lentiviral vectors.

After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring.

The plan, says the company, is to use the gene-editing method known as CRISPR to introduce extensive DNA modifications into pigs as a way of humanizing their organs so they won’t get rejected if transferred into a person. (The back of the T-shirt reads: “PS, I like my bacon extra CRISPR’ed.”)

The company is a spinout of the lab of Harvard Medical School geneticist George Church. He and cofounder Luhan Yang, who is also chief scientific officer, showed in 2015 that using gene editing, a novel and powerful way of modifying DNA inside living cells, they could eliminate viruses that lie latent in the pig’s genome.

The Polycomb Group protein EZH2 is upregulated in most prostate cancers, and its overexpression is associated with poor prognosis. In this study, the authors developed one such transgenic mouse model for conditional overexpression of Ezh2. Compounding a previously described Bmi1-transgene and Pten-deficiency prostate cancer mouse model with the Ezh2 transgene did not enhance tumour progression or drive metastasis formation. In conclusion, they here 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.

This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA.

Here, the scientists CRISPRtools platform supports loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. . In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.

In this work, scientists developed the CRISpr MEdiated REarrangement (CRISMERE) strategy, which takes advantage of the CRISPR/Cas9 system, to generate most of the desired rearrangements from a single experiment at much lower expenses and in less than 9 months. Deletions, duplications, and inversions of genomic regions as large as 24.4 Mb in rat and mouse founders were observed and germ line transmission was confirmed for fragment as large as 3.6 Mb. CRISMERE is even more powerful than anticipated it allows the scientific community to manipulate the rodent and probably other genomes in a fast and efficient manner which was not possible before.

Here, the authors report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, they report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate.

Here the scientists report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, they found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. These findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.

Here, the authors describe theirpoint of view about the CRISPR revolution for murin genetics field.

Here, the authors used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. They demonstrated that CRISPR system facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

In this study, the scientists utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. They found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.