Journal: Molecular Medicine Reports
Article Title: TALENs-mediated homozygous CCR5Δ32 mutations endow CD4+ U87 cells with resistance against HIV-1 infection
Figure Lengend Snippet: TALENs-mediated homozygous recombination. (A) T7E1 enzyme analysis of transfected CD4 + U87 cells after each transfection with CCR5-TALENs. The targeting efficiencies after first, second and third rounds of transfection were 14.80, 38.20 and 50.04%, respectively. M, 100 bp marker; wild-type, wild-type cells; lanes 1–3, transfected cells after the first, second and third rounds of transfections. (B) PCR of CCR5Δ32/Δ1 genotype cells. M, 100 bp marker; lane 1, prior to transfection; lane 2, after CCR5-TALENs transfection; lane 3, after CCR5Δ1-TALENs transfection; the CCR5Δ32 band (168 bp) was 2.87-times brighter than the CCR5Δ1 band; lane 4, wild-type cells (negative control); lane 5, CCR5Δ32 donor DNA (positive control). (C) Sequencing of wild-type CD4 + U87 cells and those with CCR5Δ32/Δ1 mutations. (D) PCR of 34 single-cell cultured clones of CD4 + U87 cells with CCR5Δ32/Δ1 mutations post-transfection with CCR5Δ1-TALENs. The CCR5Δ32 band was observed in all clones; however, as a single band, it was only seen in clones 16, 18 and 25 post-transfection with CCR5Δ1-TALENs. M, 100 bp marker; lanes 1–34, single-cell cultured cells post-transfection with CCR5Δ1-TALENs; wild-type, wild-type cells (negative control); donor, CCR5Δ32 donor DNA (positive control). (E) Gene sequencing of clones 16, 18 and 25. CCR5, C-C chemokine receptor type 5; CD4, cluster of differentiation 4; PCR, polymerase chain reaction; T7E1, T7 endonuclease 1; TALENs, transcription activator-like effector nucleases.
Article Snippet: After three rounds of transfection, DNA was extracted using the Blood Genomic DNA Extraction Mini kit (Tiangen Biotech Co., Ltd., Beijing, China) for T7 endonuclease 1 (T7E1) enzyme analysis, and ≥50 transfected cells were cultured individually.
Techniques: TALENs, Transfection, Marker, Polymerase Chain Reaction, Negative Control, Positive Control, Sequencing, Cell Culture, Clone Assay