Sprague Dawley Rag2-Null Rats Created from Engineered Spermatogonial Stem Cells Are Immunodeficient and Permissive to Human Xenografts

Abstract

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2 ^tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non-small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481-9. ©2018 AACR.

Fig. 1.

Disruption of Rag2 gene in rat SSCs. (A) The XTN pair targets early in the single coding exon of Rag2 gene. XTN binding sites are capitalized, the Rag2 start codon is shown in boldface font and underlined, the MseI site utilized for genotyping is marked. (B) Alignment of clones to the wild type reference sequence. Reference sequence is underlined, the XTN binding sites are shown in uppercase, mutations are shown in bold font and Rag2 start codon is in bold font and underlined. Clone E10 contains a single nucleotide deletion, clone A05 contains a two nucleotide deletion.

Fig. 2.

Expression of ZBTB16 (PLZF) in MEFs, fresh SSCs, and parental SD-WT2 SSCs kept in culture for 4.5 months (passage 16). Cells were stained with anti-PLZF antibody (red) and Hoechst 33342 (blue). (A) Fresh SSCs (passage 9) on laminin shows undifferentiated spermatogonia that express ZBTB16 (PLZF), whereas the MEFs do not. (B) Immunocytochemistry of SSC clusters at passage 16 on feeders. First column with Hoechst33342 nuclear staining shows SSCs (long arrow) and feeders (short arrow). Second column shows PLZF staining. Third column shows merged images of PLZF staining and Hoechst33342.

Fig. 3.

Genotyping pups for disruptions in Rag2 gene. The targeted locus was amplified from pups sired by the implanted male and subjected to MseI digestion. Animal 2f contained at least one allele that is resistant to MseI digestion (white arrow). The PCR product from animal 2f was TOPO cloned and sequenced which revealed this animal carries a mutant allele with a 27bp deletion.

Fig. 4.

Immunophenotyping of the SDR rat. (A) SDR thymocytes contain only 7.55% CD4+/CD8+ mature T cells (right panel), compared to 89.24% in a wild-type control (left panel). The majority of thymocytes are CD4 and CD8 double negative in the SDR rat. (B) The SDR rat spleen contains no mature B cells as demonstrated by lack of B220+/IgM+ cells (right panel), whereas the wildtype spleen contains 37.84% B220+/IgM+ mature B cells (left panel). (C, D) SDR rat spleen (C) and thymus (D) have an increased NK cell population (43.94% and 5.41%, respectively) compared to only 3.97% in the wild-type spleen (C) and less than 1% in the wild-type thymus. Left panels: wild-type rat. Right panels: SDR rat.

Fig. 5.

Subcutaneous growth of human glioblastoma U87MG cells in the SDR rat. 1×10⁶ U87MG cells resuspended in Geltrex were injected subcutaneously into SDR rats. (A) Tumor growth in two different SDR animals with images of their excised tumors. (B) Tumor volume (mm³) over time. Each line represents tumor growth in an individual rat. (C) Immunohistochemistry of anti human-mitochondria in tumor tissue and rat tissue. Brown staining demonstrates peri-nuclear localization of human-mitochondria protein in a tumor section, with (right) and without (left) hematoxylin counterstain. 40x magnification. The antibody for human mitochondria protein does not show staining in tissue from a rat that was not injected with human cells (negative control). Right panel with hematoxylin counterstain; 40× magnification; Scale bar = 100μm.

Fig. 6.

Subcutaneous tumor growth of NSCLC, ovarian and endometrial cells in SDR rats. (A) H358 cancer cells were transplanted subcutaneously in the SDR rat. Three groups of six rats received either 1×10⁶, 5×10⁶ or 1×10⁷ cells in 5mg/ml Geltrex. In comparison, 1×10⁷ H358 cells were injected in six nude and six NSG mice and growth was tracked for 60 days. Average tumor growth (mm³) over time. (B) 2×10⁶ OV81 cells resuspended in 5mg/ml Geltrex were injected subcutaneously into three female SDR rats. Graph shows three individual rat tumor volumes over time (mm³). Each line represents tumor growth in an individual rat. (C) 2×10⁶ OV185 cells resuspended in 5mg/ml Geltrex were injected subcutaneously into two female SDR rats. Graph shows two individual rat tumor volumes over time (mm³). Each line represents tumor growth in an individual rat.