Journal:

Article Title: Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in na?ve T cells

doi:

Figure Lengend Snippet: Expression of CARΔ1 and wt CAR on Tg lymphocytes. (A) CAR expression does not perturb T cell development. Lymphocytes were isolated from the spleen or thymus of either non-Tg, wt CAR Tg, or CARΔ1 Tg mice, stained with fluorescent-conjugated antibodies to CAR, CD4, CD8, and/or B220, and analyzed by flow cytometry (CD4 and CD8 expression in the spleen and thymus is shown). The percentages shown for cells in each quadrant represent the average for four sets of age- and sex-matched mice. Thymic and splenic cellularity did not differ significantly between mice of the different genotypes. Average cell number ± SD (×107) for the thymus: non-Tg, 7.7 ± 3.6; CARΔ1, 8.6 ± 4.7; and wt CAR, 7.5 ± 3.4. For the spleen: non-Tg, 9.9 ± 2.3; CARΔ1, 12.0 ± 5.6; and wt CAR, 14.1 ± 1.6. (B) The expression of CAR on lymphocyte subsets. The expression of CAR on peripheral blood T cells (B220-negative lymphocytes), B cells (B220 positive), CD4+CD8+ thymocytes, or CD4−CD8− thymocytes was determined by flow cytometry as in A. The mean value of fluorescence for the entire cell population is indicated inside each histogram. (C) The CAR Tg mRNA is specifically expressed in the thymus and peripheral lymphocytes. Total RNA was isolated from the indicated tissues from either non-TG (−) or CARΔ1 Tg mice and analyzed for the expression of the human CARΔ1 transgene by Northern blotting. Lymphocyte RNA was isolated from the spleen and lymph nodes combined. The positions of RNA size markers are indicated. The expression of 28S and 18S rRNA (methylene blue stain) is shown as a loading control.

Article Snippet: Antibodies to B220 (allophycocyanin-linked; no. 553092), CD4 (phycoerythrin-linked; no. 09005B), and CD8 (Cy-Chrome-linked; no. 553034) were purchased from PharMingen.

Techniques: Expressing, Isolation, Staining, Flow Cytometry, Fluorescence, Northern Blot

Journal:

Article Title: Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in na?ve T cells

doi:

Figure Lengend Snippet: CAR Δ1 Tg mature T cells and thymocytes are efficiently transduced by recombinant adenovirus. (A) Transduction of mature lymphocytes. Lymphocytes from CAR Tg or non-Tg mice were harvested from the spleen and lymph nodes (pooled), transduced with either AdCMV-GFP or AdUbC-GFP at a MOI of 10, cultured overnight, and stained with fluorescent-tagged antibodies to CD4 and CD8. GFP expression (displayed on the x axis; the y axis represents cell number) was monitored by flow cytometry in lymphocytes gated for the expression of either CD4, CD8, or neither (B cells). In A and B, percentages of GFP-positive cells are indicated inside each histogram. Gating for GFP-positive cells was determined based on the fluorescent intensity of mock-transduced cells. (B) Transduction of CAR Tg thymocytes. Thymocytes from CARΔ1 Tg and non-Tg mice were transduced with AdUbC-GFP at a MOI of 5 as described in A. GFP expression was determined in thymocytes gated for the expression of both CD4 and CD8 or neither (CD4−CD8− thymocytes). (C) Dose-dependent gene expression. Lymphocytes from CARΔ1 Tg mice were transduced with AdUbC-GFP at the indicated MOIs as described in A. The mean value of GFP intensity for transduced T cells is plotted relative to the MOI. (D) T cell activation potentiates CAR-mediated adenoviral delivery. Peripheral lymphocytes (DO11.10 TCR and CARΔ1 double Tg) were harvested and either immediately transduced with AdUbC-GFP at a MOI of 1 (Unactiv.), or cultured in RP10 together with 5 μg/ml OVA (ISQAVHAAHAEINEAGR) or 4 μg/ml Con A for 2 days before transduction with AdUbC-GFP at a MOI of one. Twenty-four hours after transduction, the cells were harvested, stained with anti-B220, and analyzed for GFP expression in lymphocytes by flow cytometry. The upper and lower quadrants represent B cells and T cells, respectively. The percentages in each quadrant are indicated. (E) Manipulation of gene expression in T cells in vivo. Lymphocytes from CARΔ1 Tg mice were harvested and transduced with AdUbC-GFP at a MOI of 10 ex vivo as described in A. Transduced cells (or mock-transduced cells) were washed twice with PBS and immediately transferred into recipient FvB mice by subocular injection; 1, 3 and 5 days after transfer, splenocytes from recipient mice were harvested and stained with antibodies to CAR and B220. The expression of CAR and GFP was determined in T cells (B220 negative) by flow cytometry.

Article Snippet: Antibodies to B220 (allophycocyanin-linked; no. 553092), CD4 (phycoerythrin-linked; no. 09005B), and CD8 (Cy-Chrome-linked; no. 553034) were purchased from PharMingen.

Techniques: Recombinant, Transduction, Cell Culture, Staining, Expressing, Flow Cytometry, Activation Assay, In Vivo, Ex Vivo, Injection