FIGURE 3 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

NoGlow mice express eGFP and Luciferase without background fluorescence and bioluminescence. A, Diagram of the NoGlow construct. LoxP-flanked stop site prevents expression of the NoGlow construct in the absence of Cre recombinase. However, in the presence of Cre the stop site is recombined to yield a single transcript encoding rtTA3 and mutant GFP/Luc driven by the CAG promoter. B, Chimeric founder NoGlow animals were crossed to CMV (full-body) Cre animals to test activity and expression of the construct. Representative bioluminescence imaging of female C57Bl/6, CAG Luc-GFP, and F1 CMV Cre NoGlow littermates with or without the NoGlow construct reveal no background bioluminescence in CMV Cre NoGlow+ animals. C, Bioluminescent imaging of representative lungs/livers shows no background bioluminescence in NoGlow+ mice. D, Chromogenic Luciferase staining in livers from NoGlow+ and control mice confirms luciferase protein expression despite the lack of bioluminescence in B and C. E, Representative fluorescent imaging of male C57Bl/6, CAG Luc-GFP, and F1 CMV Cre NoGlow animals shows no background fluorescence in CMV Cre NoGlow+ mice, while immunofluorescent staining (F) for GFP in liver sections from CMV Cre NoGlow+ animals confirms that GFP protein can be detected upon antibody staining.</p

FIGURE 7 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Summary of tumor behavior in differentially tolerant animal models. A, Xenoantigen-bearing tumor cells implanted orthotopically into immune-deficient mice typically grow and metastasize as expected and can be easily observed with fluorescence/bioluminescence. B, Conversely, in immune-competent animals the presence of foreign immunogens often results in complete rejection of the primary tumor, or severely restricts expression of immunogenic antigens and limits the utility of reporter proteins. C, However, NoGlow mice are tolerant to foreign GFP, Luciferase, and rtTA, thus allowing primary tumor growth and de novo metastasis of xenoantigen-expressing tumor cells.</p

FIGURE 2 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Mutant GFP and luciferase induce immune responses comparable with wild-type (WT) proteins. A, WT male Balb/c mice were vaccinated with Ad encoding WT or mutant copies of GFP (ΔT64; n = 5/group) and serum antibody responses were measured after 3 weeks. Mice showed equivalent anti-GFP responses whether vaccinated with WT or mutant GFP. B, WT female C57Bl/6 mice were vaccinated with Ad-GFP-Luc WT or Ad-Luc mutant (G315A; n = 5/group) and serum antibody responses were measured after 3 weeks revealing similar levels of anti-Luc antibodies. P values for A, B are at 1:50 dilution by two-way ANOVA with Tukey multiple comparisons test. C, Male WT Balb/c mice were vaccinated with Ad encoding WT or mutant GFP (ΔT64) or Luc (H245R) and anti-GFP or -Luc T-cell responses in the spleens were detected after 3 weeks via ELISpot. Both WT and mutant forms of GFP and Luc were able to elicit robust T-cell responses compared with mice naïve for the respective antigen. P values were determined by two-way ANOVA with Tukey post hoc analysis. D, E0771 GFP-Luc cells were implanted in MFPs of female WT C57Bl/6 mice and were vaccinated with Ad encoding WT GFP, mutant GFP (ΔT64), WT Luc, mutant Luc (ΔH245), or LacZ control (n = 5 per group) after three days. Compared with LacZ control vaccination, GFP WT/mutant, or Luc WT/mutant vaccines resulted in a comparable reduction in tumor growth. P values are at time of euthanasia by two-way ANOVA with Tukey multiple comparisons test. All P values represent mean ± SEM.</p

Figure S2 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Validating mutant GFP and Luc activity in Adenovirus vaccines</p

Figure S1 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Additional E0771 tumor growth in WT, GH, and CAG Luc-GFP mice; MMTV CAG HER2 Ad-HER2 vaccination T cell responses.</p

FIGURE 6 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

NoGlow mice reveal unappreciated metastatic dynamics independent of the primary tumor. A, Tumor growth of triple transgenic (3 ×) B16-F10 cells (105) after subcutaneous implantation into the flank of WT C57Bl/6 (n = 2), CAG-driven Full-body WT GFP/Luc expressing mice (n = 3), CMV cre littermates positive (n = 8) or negative (n = 5) for the NoGlow construct, or SCID beige (n = 5) mice. B, Bioluminescence imaging of lungs at the time of euthanasia for each mouse from A. Robust signal is detected only in NoGlow+ lungs. *Indicates no detectable primary tumor at the end of experiment. C, Bioluminescence intensity of individual lungs from B. NoGlow+ lungs contained significantly more positive Luc signal than SCID, WT, or NoGlow− animals. D, Plot of tumor volume at time of euthanasia (x) by total lung radiance (y) for individual animals from A. P values were determined by one-way ANOVA with Tukey correction. All P values represent mean ± SEM.</p

FIGURE 5 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Tumor cells expressing GFP, rtTA, and Luciferase successfully engraft in NoGlow mice. A, Diagram of triple-transgenic (3 ×) E0771 cells. GFP and rtTA are constitutively expressed and Luc is induced with the addition of doxycycline. B, 3 × E0771 (106) were implanted into the mammary fat pad (MFP) of wild-type (WT) C57Bl/6 (n = 7), CAG-driven Full-body WT GFP/Luc expressing mice (n = 7), CMV cre littermates positive (n = 6) or negative (n = 7) for the NoGlow construct, or SCID beige (n = 4) mice. C, Overall (top) and individual (bottom) tumor growth in each group. No evidence of tumors was observed in Full-body GFP-Luc, NoGlow−, or C57Bl/6 animals by the end of experiment. D, Representative bioluminescence imaging of animals pre (left) or post (right) doxycycline diet introduction. Bottom, Luc was induced in SCID mice but to a lesser degree compared with NoGlow+ mice. E, Representative GFP imaging upon euthanasia of Noglow+, SCID, and Full-body GFP-Luc mice. Left, concentrated GFP signal in tumors of NoGlow+ and SCID compared with Full-body GFP-Luc mice. Right, visceral GFP signal only in Full-body GFP-Luc mice. F, Representative lungs of NoGlow+, NoGlow−, Full-body GFP-Luc, or SCID mice upon euthanasia. Lung metastasis can be visualized in NoGlow+ and SCID animals.</p

Table S2 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Peptides used for ELISPOTs</p

Figure S3 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Validating the NoGlow construct in vitro</p

Figure S4 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Confirming triple-transgenic GFP rtTA-Luc construct in E0771 cells</p