Fig 1.
MLL, AF4, and AF9 TALEN design.
(A, C) TALEN pairs (bold sequence) were designed to target known translocation break points (underlined) within the MLL, AF4, and AF9 genes. (B, D) Following nucleofection into K562 cells, cutting by each TALEN pair was measured using the surveyor assay, which detects indels within the target region that result from imprecise repair of a DNA double strand break. Asterisks denote cleaved PCR products.
Fig 2.
Co-expression of MLL and AF4 or AF9 TALENs in K562 cells induces MLL translocations.
(A, B) MLL and AF4 or AF9 TALENs were transiently expressed in K562 cells. Genomic DNA was isolated at the indicated time point and used for PCR analysis for MLL translocations. (C, D) PCR products were isolated and sequenced to confirm translocation products. Data shown are a composite alignment of PCR products from multiple experiments showing a variety of distinct translocations. Underlines denote TALEN binding sites.
Fig 3.
Co-expression of MLL and AF4 or AF9 TALENs in primary human CD34+ cells induces MLL translocations.
(A, B) Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with MLL and AF4 or AF9 TALENs. Genomic DNA was isolated at the indicated time point and used for PCR analysis for MLL translocations. (C, D) PCR products were isolated and sequenced to confirm translocation products. Nucleotide sequences shown are a composite alignment of PCR products from multiple experiments showing a variety of distinct translocations. Underlines denote TALEN binding sites.
Fig 4.
Survival advantage for CD34+ cells containing an MLL-AF4 translocation.
Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with MLL and AF4 TALENs. The population was immediately divided into 20 subcultures (50,000 cells per well), which were maintained over time. Genomic DNA was isolated at the indicated time points and used for semi-quantitative PCR analysis for MLL translocations (A) and qPCR for the MLL-AF4 translocation (B). (-- indicates subcultures nucleofected with GFP as negative control)
Fig 5.
FISH analysis shows MLL translocations in human CD34+ cells induced by MLL and AF4 TALENs.
(A-D) FISH analysis using an MLL break apart probe was performed on cells from subcultures 9 (A, B) and 20 (C) as well as a negative control culture (D) to evaluate the presence of MLL chromosomal translocations. Two hundred cells were analyzed for each sample. Representative images are shown. Red arrows indicate cells positive for an MLL translocation. Panel B shows a cell from subculture 9 in metaphase that was noted to have an MLL translocation.
Fig 6.
CD34+ cells nucleofected with MLL and AF4 or AF9 TALENs display enhanced replating potential in semi-solid medium.
Colony forming assays were performed to assess the effect of MLL and AF4 or AF9 TALENs on the replating efficiency of CD34+ cells in semi-solid medium. The bars in Panel A represent the mean number of colonies generated per 104 seeded cells. Panel B shows the morphology of secondary colonies generated by CD34+ cells nucleofected with MLL and AF4 or AF9 TALENs: diffuse colony representing ~80% (left) and compact colony representing ~20% (right) of all colonies.