Animal Models - GEG Tech top picks

Around 12% of men are diagnosed with prostate cancer during their lifetime. When prostate cancer is confined to the primary tumor, survival is close to 100%. When the cancer spreads or metastasizes, the patient's chances of survival drop to less than 40%. In a recent study, published on September 23 in Cancer Discovery, scientists have mapped the complex routes taken by metastatic prostate cancer cells as they move through the body by creating a new mouse model called EvoCaP and using CRISPR and a barcode system. They observed that while the primary tumor contained many prostate cancer cells, most metastases began with a small number of aggressive clones emerging from the tumor and moving to the bones, liver, lungs and lymph nodes. They also observed that once most cancer cells spread to an organ, they were likely to stay there rather than spread to another area, with just a few closely related cells instigating additional spread. A better understanding of prostate cancer metastasis opens the way to better treatments.

Genomic studies of cancer patients have revealed thousands of mutations linked to tumor development. In an advance that could help scientists make a dent in that long list of unexplored mutations, MIT researchers designed their new mouse models by engineering the gene for the prime editor enzyme into the germline cells of the mice. The encoded prime editor enzyme allows cells to copy an RNA sequence into DNA that is incorporated into the genome. However, the prime editor gene remains silent until activated by the delivery of a specific protein called Cre recombinase. Since the prime editing system is installed in the mouse genome, researchers can initiate tumor growth by injecting Cre recombinase into the tissue where they want a cancer mutation to be expressed, along with a guide RNA that directs Cas9 nickase to make a specific edit in the cells' genome. The RNA guide can be designed to induce single DNA base substitutions, deletions, or additions in a specified gene, allowing the researchers to create any cancer mutation they wish. Using this technique, the researchers have created models of several different mutations of the cancer-causing gene Kras, in different organs. Such models could help researchers identify and test new drugs that target these mutations. 

Biologists have embraced CRISPR’s ability to quickly and cheaply modify the genomes of popular model organisms, such as mice, fruit flies and monkeys. Now they are trying the tool on more-exotic species, many of which have never been reared in a lab or had their genomes analysed. “We finally are ready to start expanding what we call a model organism,” says Tessa Montague, a molecular biologist at Columbia University in New York City. 

But the practical challenges of breeding and maintaining unconventional lab animals persist.

Knock-in mice with precise insertions are efficiently generated through delivery of CRISPR–Cas9 to two-cell embryos.

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation.

Here, the scientists confirmed the risk of cross-species transmission of porcine endogenous retroviruses (PERVs), and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, they inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. This study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.

In the present study, The authors applied CRISPR/Cas9 system to target the C3 gene in porcine fetal fibroblasts. Their results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. 

Modeling human disease has proven to be a challenge for the scientific community. For years, generating an animal model was complicated and restricted to very few species. With the rise of CRISPR/Cas9, it is now possible to generate more or less any animal model. In this review, we will show how this technology is and will change our way to obtain relevant disease animal models and how it should impact human health.

Genome editing in pigs has tremendous practical applications for biomedicine. The advent of genome editing technology, with its use of site-specific nucleases has popularized targeted zygote genome editing via one-step microinjection in several mammalian species. Here, the authors review methods to optimize the developmental competence of genome-edited porcine embryos and strategies to improve the zygote genome-editing efficiency in pigs

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Although the authors reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required and exposed in this article. The findings of this study demonstrate for the first time that carboxylated ε-poly-L-lysine  is a promising cryoprotective agent for further improvements in the vitrification of oocytes and embryos in mammalian species.

Here, the scientists report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. Their procedure combining the CRISPR/Cas9 system and in utero electroporation and they showed that it is an effective and rapid approach to achieve brain-specific gene knockout in vivo.

In a recent study, researchers used a breed of humanized Yucatan miniature pigs as a porcine donor to develop kidney transplants without the three major glycans (cytidine monophospho-Nacetylneuraminic acid hydroxylase, glycoprotein α-galactosyltransferase 1 and β-1,4-N-acetylgalactosaminyltransferase 2) and with porcine retroviral genes inactivated. A Cas9-associated single-guide ribonucleic acid guide design with clustered regularly interspaced short palindromic repeats (CRISPR) was used to target genes encoding the three major glycans expressed on the porcine cell surface. The grafts were also designed to overexpress human transgenes. These kidney grafts were transplanted into Macaca fascicularis or cynomolgus monkeys, which were used as non-human primate models to test the efficacy and safety of the kidney grafts. Kidneys from which only the three porcine glycan genes had been eliminated showed poor graft survival after transplantation into the non-human primate model. In comparison, those modified to eliminate all three glycan genes and overexpress human transgenes showed significantly better survival in cynomolgus monkeys.

Studies of diseases that affect the human brain are generally based on animal models that cannot replicate the complexity of human neuropathies. Consequently, these methodologies often fail when applied in a clinical setting with patients. In this context, the discoveries of cell reprogramming techniques to generate human neuron cultures from skin cells have revolutionized the study and development of innovative therapies in neuroscience. A study published in the journal Stem Cells Reports reveals that this cell reprogramming methodology allows the creation of neural networks that mimic unique features of human cells with temporal dynamics reminiscent of human brain development. Thus, cellular models based on reprogrammed human cells could stimulate the development of new effective therapies in the fight against neuropathies and, at the same time, reduce the use of experimental animals in the laboratory. In addition, cell reprogramming based on the induction of human pluripotent stem cells would allow the generation of patient-specific models and, using gene editing tools such as the CRISPR/Cas9 technique, it would be possible to obtain control cells in which the mutation responsible for the pathology is corrected. 

Australia has approved the use of CRISPR gene editing tool on plants and animals without the oversight of a governmental body. The controversial move has been called a 'middle ground' compared to regulations on other countries.

For the first time, scientists have performed prenatal gene editing to prevent a lethal metabolic disorder in laboratory animals, offering the potential to treat human congenital diseases before birth. Published today in Nature Medicine, research from Children’s Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania offers proof-of-concept for prenatal use of a sophisticated, low-toxicity tool that efficiently edits DNA building blocks in disease-causing genes.

In the present study, the CRISPR/Cas system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at 38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer (SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth. However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Here, the scientists describe the generation of transgenic, inducible CRISPR-based mouse systems to engineer and study recurrent colon cancer-associated EIF3E-RSPO2 and PTPRK-RSPO3 chromosome rearrangements in vivo. 

Genome editing for spermatogonial stem cells (SSCs) still remains a big challenge mainly due to low efficiency and complexity of currently available techniques. T

Here the authors describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

Using the gene-editing system known as CRISPR, MIT researchers have shown in mice that they can generate colon tumors that very closely resemble human tumors. This advance should help scientists learn more about how the disease progresses and allow them to test new therapies.