human epithelial carcinoma cell line hela  (ATCC)

Journal: Nucleic Acids Research

Article Title: rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination

doi: 10.1093/nar/gkae401

Figure Lengend Snippet: The Y704T mutant led to transcriptional defects of rAAV vector-delivered transgene in vitro . (A, B) HEK293 cells were transduced with 19 rAAV2-Y704 mutants and rAAV2-WT vector at 100 000 vg/cell for 72 h. Fold change (FC) of ( A ) gfp expression, and ( B ) mRNA copy numbers were detected by microscopy and qPCR, respectively. Data was normalized by WT. A: Y704A, C: Y704C, D: Y704D, E: Y704E, F: Y704F, G: Y704G, H: Y704H, I: Y704I, K: Y704K, L: Y704L, M: Y704M, N: Y704N, P: Y704P, Q: Y704Q, R: Y704R, S: Y704S, T: Y704T, V: Y704V, W: Y704W, WT: Y704. ( C ) HEK293 cells were transduced with rAAV2-WT, -Y704T or -Y704F-CMV- gfp vectors at indicated MOIs for 72 h. gfp expression was detected by flow cytometry. ( D ) HEK293, Huh7 and HeLa cells were transduced with rAAV2-WT, -Y704T or -Y704F-CMV- gfp vectors at 10 000 vg/cell for 72 h. Vector mRNA was quantified by qPCR assays. ( E ) Relative gfp mRNA expression in HEK293, Huh7 and HeLa cells following transduction with rAAV3 (left) and rAAV6 (right)-WT or -Y705T-CMV- gfp vectors. ( F ) Luciferase expression in HeLa cells transduced with single-stranded rAAV-CMVp- fluc vectors (left) and gfp expression in HeLa cells transduced with self-complementary rAAV-CMVp- gfp vectors (Right). rAAV2-WT: 2WT, rAAV2-Y704T: 2T, rAAV2-Y704F: 2F, rAAV3-Y705T: 3T, rAAV3-Y705F: 3F, rAAV6-Y705T: 6T, rAAV6-Y705F: 6F. ( G ) Vector genomes (VG) in HEK293 cells following transduction with rAAV-CMV- gfp vectors. Scale bar: 200 μm. Data are presented as mean ± standard deviations. Differences of two groups were analyzed by two-tailed unpaired t test, one-way ANOVA analysis with Dunnett's multiple comparisons test was used in the case of three groups. * P < 0.05, ** P < 0.01, *** P < 0.005. ns statistically not significant.

Article Snippet: The human embryonic kidney cell line 293 (HEK293), the epithelial carcinoma cell line HeLa, and HeLaRC (a stable derivative of HeLa cells harboring one to two rep-cap gene copies per cell) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Mutagenesis, Plasmid Preparation, In Vitro, Transduction, Expressing, Microscopy, Flow Cytometry, Luciferase, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination

doi: 10.1093/nar/gkae401

Figure Lengend Snippet: The late infectious pathways of rAAV-Y704T mutants. ( A ) Confirmation of nuclear (Nuc) and cytoplasmic (Cyt) fraction purity. GAPDH: cytoplasmic marker, LaminB1: nuclear marker. ( B ) DNA distribution of rAAV-CMV- gfp in nuclear and cytoplasmic fraction. ( C ) The percentage of uncoated (DNase-sensitive) genome in the nucleus was determined by splitting the nuclear fraction in half and treating one half with Benzonase before viral DNA isolation to remove any uncoated genome. The other half was used to determine the total number of genomes in the nucleus. ( D ) HeLa cells were transduced with variant rAAV-CMV- fluc vectors with BRD4770 or CPI-455. The expression of firefly luciferase was measured at 72 h post-transduction. ( E ) HeLa cells were transduced with variant rAAV-CMV- fluc vectors with 5-AZ, the expression of firefly luciferase was measured at 72 h post-transduction. ( F ) HeLa cells were transduced with variant rAAV-CMV- fluc vectors with DBeQ, the expression of firefly luciferase was measured at 72 h post-transduction. ( G ) The mice were treated with different concentration of DBeQ by intravenous injection, the rAAV6-Y705T-CMV- fluc was injected into the tibialis anterior muscles (TAs) of mice at the same time. The mRNA of fluc in TAs was determined by qPCR. Data presented are mean ± standard deviations. Differences of two groups were analyzed by two-tailed unpaired t test, one-way ANOVA analysis with Dunnett's multiple comparisons test was used in the case of three or more groups. * P < 0.05, ** P < 0.01, *** P < 0.005. ns statistically not significant.

Article Snippet: The human embryonic kidney cell line 293 (HEK293), the epithelial carcinoma cell line HeLa, and HeLaRC (a stable derivative of HeLa cells harboring one to two rep-cap gene copies per cell) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Marker, DNA Extraction, Transduction, Variant Assay, Expressing, Luciferase, Concentration Assay, Injection, Muscles, Two Tailed Test