There are multiple ways to create somatic mutations in rodents. Initial models were built on RCAS (replication-competent avian sarcoma-leukosis) vectors, which required mice with germline TVA (tumor virus A) retroviral receptors linked to cell-type-specific promoters [9, 10, 12, 13, 16]. Canoll et al. used a construct with replication-deficient retrovirus plasmids and vesicular stomatitis virus G (VSV-G) coats to somatically introduce oncogenes into rodents [8, 14]. The Sleeping Beauty [6] model employs the SB transposase enzyme to transfer genetic material from plasmids into host chromosomes after injection into neonatal mice lateral cerebral ventricles. Using various combinations of human oncogenes and inhibitors of tumor suppressor function, they were able to reliably induce spontaneous tumors resembling human GBM [17]. Finally, lentivirus vectors have been used with increasing frequency to express oncogenes (HRAS, KRAS, PDGF, AKT) and to knock down tumor suppressors (p53 and INK4) [11, 15, 18]. The lentivirus vector system is ideal due to the ability of LV vectors to transduce both dividing and nondividing cells and encode large (up to 9.7 kb) DNA sequences [19–21].

Models utilizing lentivirus to induce GBM often take advantage of the Cre-Lox system [11, 15]. One recent report used a lentiviral vector encoding floxed HRAS and AKT oncogenes in GFAP-Cre transgenic mice (with or without p53 heterozygosity) [15]. The combination of these oncogenes was sufficient to cause high-grade gliomas when injected into neurogenic areas (hippocampus and subventricular zones), and the loss of p53 increased their incidence and decreased their latency. In another report, a GFAP-Cre lentivirus was injected into transgenic mice carrying various combinations of floxed genes [11]. In this model, activated KRAS and loss of INK4a/ARF were sufficient to cause high-grade gliomas, and the additional loss of p53 caused higher-grade tumors to form more rapidly. The latter model was also used as a screen for therapeutic interventions, in which temozolomide was tested and shown to be effective [11].

Lentivirus-mediated tumor induction in rats is more effective than in mice, often not requiring mutations in tumor suppressor pathways. Intracranial injection of a retrovirus expressing PDGF-β has induced GBM with complete penetrance and short latency in rats [8]. In recent experiments, injection of lentiviral vectors expressing PDGF-β and HRAS-V12 respectively induced GBM in Sprague-Dawley rats with complete penetrance and latency ranging from 27 to 60 days [18]. The resulting glioblastomas demonstrated the nuclear atypia, mitoses, endothelial proliferation, and necrosis that constitute the pathologic hallmarks of the human disease (Fig. 1). Injection of lentiviral vectors expressing HRAS-V12 alone produced slower growing gliomas with high penetrance but did not cause animal morbidity until 6-month post-virus delivery. In addition, this model allowed us to test a gene therapy strategy for glioblastoma, i.e., adenovirus vectors encoding herpes simplex type 1 thymidine kinase (b), which induces tumor cell death when used in combination with ganciclovir (GCV), thus adding relevant knowledge related to a therapeutic modality recently tested in a Phase 3 clinical trial for GBM in Europe [22–24]. These models demonstrate the versatility and ease of modifying the genetic profile in situ with observable, meaningful results.

The protocols below will address (Sect. 3.2, step 1) lentiviral vector production, (Sect. 3.2, step 2) intracranial lentiviral injection in rats, and (Sect. 3.2, step 3) testing of a conditional cytotoxic gene therapy, i.e., herpes simplex virus type I thymidine kinase (HSV1-TK) encoded within a first-generation adenovirus vector (Ad-TK) in combination with systemic delivery of ganciclovir (GCV), in the LV-induced endogenous GBM model.