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Genetically Engineered Mouse Models of Gliomas: Technological Developments for Translational Discoveries.

ABSTRACT: The most common brain tumours, gliomas, have significant morbidity. Detailed biological and genetic understanding of these tumours is needed in order to devise effective, rational therapies. In an era generating unprecedented quantities of genomic sequencing data from human cancers, complementary methods of deciphering the underlying functional cancer genes and mechanisms are becoming even more important. Genetically engineered mouse models of gliomas have provided a platform for investigating the molecular underpinning of this complex disease, and new tools for such models are emerging that are enabling us to answer the most important questions in the field. Here, I discuss improvements to genome engineering technologies that have led to more faithful mouse models resembling human gliomas, including new cre/LoxP transgenic lines that allow more accurate cell targeting of genetic recombination, Sleeping Beauty and piggyBac transposons for the integration of transgenes and genetic screens, and CRISPR-cas9 for generating genetic knockout and functional screens. Applications of these technologies are providing novel insights into the functional genetic drivers of gliomagenesis, how these genes cooperate with one another, and the potential cells-of-origin of gliomas, knowledge of which is critical to the development of targeted treatments for patients in the clinic.

SUBMITTER: Noorani I

PROVIDER: S-EPMC6770673 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Genetically Engineered Mouse Models of Gliomas: Technological Developments for Translational Discoveries.

Noorani Imran I

Cancers 20190909 9

The most common brain tumours, gliomas, have significant morbidity. Detailed biological and genetic understanding of these tumours is needed in order to devise effective, rational therapies. In an era generating unprecedented quantities of genomic sequencing data from human cancers, complementary methods of deciphering the underlying functional cancer genes and mechanisms are becoming even more important. Genetically engineered mouse models of gliomas have provided a platform for investigating t ...[more]

PMID: 31505839

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Project description:Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.

Dataset Information

Genetically Engineered Mouse Models of Gliomas: Technological Developments for Translational Discoveries.

Publications

Genetically Engineered Mouse Models of Gliomas: Technological Developments for Translational Discoveries.

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