Rodent models of tumours of the central nervous system

Abstract

Modelling of human diseases is an essential component of biomedical research, to understand their pathogenesis and ultimately, develop therapeutic approaches. Here, we will describe models of tumours of the central nervous system, with focus on intrinsic CNS tumours. Model systems for brain tumours were established as early as the 1920s, using chemical carcinogenesis, and a systematic analysis of different carcinogens, with a more refined histological analysis followed in the 1950s and 1960s. Alternative approaches at the time used retroviral carcinogenesis, allowing a more topical, organ-centred delivery. Most of the neoplasms arising from this approach were high-grade gliomas. Whilst these experimental approaches did not directly demonstrate a cell of origin, the localisation and growth pattern of the tumours already pointed to an origin in the neurogenic zones of the brain. In the 1980s, expression of oncogenes in transgenic models allowed a more targeted approach by expressing the transgene under tissue-specific promoters, whilst the constitutive inactivation of tumour suppressor genes ('knock out')-often resulted in embryonic lethality. This limitation was elegantly solved by engineering the Cre-lox system, allowing for a promoter-specific, and often also time-controlled gene inactivation. More recently, the use of the CRISPR Cas9 technology has significantly increased experimental flexibility of gene expression or gene inactivation and thus added increased value of rodent models for the study of pathogenesis and establishing preclinical models.

Keywords: CRISPR Cas9 system; Cre‐lox system; brain tumour; genetically modified mouse model; glioblastoma; medulloblastoma.

Fig. 1

Brain tumour models: chemical, viral and constitutive transgenic oncogenesis: The left column shows the experimental approach, the centre column the events on cellular level and the right column the events or modifications on genomic level. (A) Schematic view of the layout with model system, cellular events and genomic events. (B) Chemical systemic carcinogenesis. The organisms used for this experimental approach were mostly rats, and less commonly mice, and other rodents. Organotropic action depends on the combination of species, their genetic background and the carcinogen. On the cellular level, carcinogens are resolved by the cell and acts in the nucleus. On genomic level, the carcinogen generates point mutations. (C) Viral carcinogenesis. The model organisms were mostly hamsters, rats, mice, and other rodents. The organotropic action depends on the combination of species, their genetic background and the virus strain. On a cellular level, the virus docks onto cells using an existing receptor and replicates using the cell's machinery. On genomic level, retroviruses integrate into the genome and disrupt and potentially activate endogenous genes. In the case of RSV, the retrovirus expresses the oncogenic v‐src kinase. (D) Transgenic expression of an oncogene. This experimental approach was predominantly done in mice. The organotropic or cell specific action of the transgene depends on the promoter that drives the expression of the oncogenic protein. On a cellular level, whilst being integrated in all cells of the organism, the transgene is expressed only in cells in which the promoter is active. On genomic level, transgenes randomly integrate into the genome. The expression pattern can slightly vary depending on the integration site. (E) Gene knockout, using homologous recombination in embryonic stem cells. The gene inactivation affects all cells in the organism. It was a fundamental experimental approach to understand the function of many genes, however the targeted deletion of a majority of tumour suppressor genes, such as RB, PTEN, or APC led to embryonic lethality, and the system was very soon replaced by the refined tissue‐specific Cre‐lox system.

Fig. 2

Brain tumour models, the RCAS‐TVA system: the only organism used in this setup were mice. The organotropic or cell specific action depends on the promoter that drives the expression of the receptor. The oncogenic action depends on the engineering of the virus. On a cellular level, the mechanism of action is a transgenic expression of a virus receptor on a desired cell type, in most brain tumour models this were GFAP or Nestin promoters. The virus is genetically engineered to match the receptor. On genomic level, the transgene expression the virus receptor is integrated in all cells of the genome, but only cells in which the promoter is active (for example astrocytes for the GFAP‐TVA transgene), express the receptor. The virus is injected into the transgenic mouse, and docks onto the receptor, determining the delivery of oncogenes or, in modified experimental setup, CRISPR Cas9 constructs or Cre‐recombinase.

Fig. 3

Brain tumour models, the Cre‐lox system: (A) One mouse line is generated to express a Cre‐recombinase. The organotropic or cell specific action depends on the promoter that drives Cre expression. A second mouse line is generated with loxP recognition sites flanking a gene of interest (‘floxed’), and in the context of brain tumour modelling, these were typically tumour suppressor genes such as p53, Rb, Pten. On cellular level, the Cre transgene is integrated in all cells of the organism, but are expressed only in cells (transiently or permanently) in which the corresponding promoter is active. In this example, only GFAP‐expressing cells are targeted, i.e. the tumour suppressor gene is recombined only in these cells, notably this can occur also transiently during development. On genomic level, the transgenically expressed Cre‐recombinase recognises pairs of loxP recognition sequences, and through the formation of a loop, the DNA stretch between two loxP sites is removed. One loxP site remains in the genome. (B) A modified approach (used to target specific regions of cell types that cannot be easily targeted by transgene expression) uses a conditional knockout mouse (‘floxed mouse’), into which a virus expressing Cre‐recombinase is injected in the targeted fashion.

Fig. 4

Brain tumour models, CRISPR Cas9 system: somatic mutagenesis using the CRISPR Cas9 technology can be used in different methodological approaches, involving two (or more) components. One component comprises the delivery of a single guide RNA (sgRNA), leading to the introduction of a deletion in the desired target genes. The sgRNA directs the Cas9 nuclease to a complementary sequence in the genome where Cas9 induces a double‐strand break. Cas9 can either be expressed via delivery by a viral vector, or expressed by the host, either constitutively, or conditionally by Cre‐mediated expression in the Rosa26 lox‐stop‐lox model. The Cre‐expressing mouse is intercrossed with the Cas9 Rosa26 LSL mouse, resulting in the expression of Cas9 in the desired cell population of the CNS. This mouse is then injected with a virus expressing sgRNA, resulting in a tissue‐specific inactivation of the desired target genes. (A) Intercrossing the mouse line expressing Cre‐recombinase under a control of a cell‐ or tissue‐specific promoter with the Cas9 Rosa26 LSL mouse results in the Cas9 expression in the desired target cell population (B). Subsequently, a viral construct expressing guide RNA (C) is delivered to the brain (D), where it disrupts the gene of interest in Cas9 expressing cells. Alternatively, the sgRNA construct can be engineered to also express Cre‐recombinase (D) and delivered directly into a Cas9 Rosa 26 LSL mouse (E). The CRISPR Cas9 system can also be combined with the RCAS‐TVA or the sleeping beauty/PiggyBAC system.

Fig. 5

Brain tumour models, transposon‐transposase system (Sleeping Beauty, PiggyBAC). (A) Transgenic mouse lines expressing Cre‐recombinase are intercrossed with the Rosa26 lox‐stop‐lox conditional mouse (B), which is starts expressing piggyBAC transposases upon Cre‐mediated recombination. A third mouse line (C) expresses the transposons, which can be mobilised with the piggyBAC (PB) or the sleeping beauty (SB) transposases, and these can be engineered to cause gain or loss of function. SA, splice acceptor; SD, splice donor; PBase, piggyBac transposase; CAG, CAG promoter. PB, both piggyBac; SB, Sleeping Beauty (Adapted from [208]).