Co-expression of <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs in primary human CD34+ cells induces <i>MLL</i> translocations.

<p>(A, B) Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs. Genomic DNA was isolated at the indicated time point and used for PCR analysis for <i>MLL</i> 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.</p

<i>MLL</i>, <i>AF4</i>, and <i>AF9</i> TALEN design.

<p>(A, C) TALEN pairs (bold sequence) were designed to target known translocation break points (underlined) within the <i>MLL</i>, <i>AF4</i>, and <i>AF9</i> 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.</p

Visualization of ZFN-induced DSBs by sensitized 53BP1 foci formation assay.

<p>(A) Representative cells for each experimental condition after 53BP1 staining using indicated amounts of transfected DNA of each nuclease in the presence or absence of drug. 53BP1 foci are seen in red, 4,6-diamidino-2-phenylindole staining in blue, and GFP-positive cells in green. The foci were counted in transfected cells that were GFP-positive. “Untransfected” shows the background staining for foci in these cells, GFP indicates transfection of GFP alone, Sce serves as a negative control for ZFN-induced foci formation, and Caspase Activated DNAse (CAD) serves as a positive control for 53BP1 foci formation. ((−)) indicates no Shield1 treatment and ((+)) indicates 1000 nM Shield1 treatment for 24 hours after transfection. (B) The average number of 53BP1 foci per transfected cell in Ku80^−/− murine 3T3 cells for each experimental condition in (A).</p

Co-expression of <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs in K562 cells induces <i>MLL</i> translocations.

<p>(A, B) <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs were transiently expressed in K562 cells. Genomic DNA was isolated at the indicated time point and used for PCR analysis for <i>MLL</i> 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.</p

FISH analysis shows <i>MLL</i> translocations in human CD34+ cells induced by <i>MLL</i> and <i>AF4</i> TALENs.

<p>(A-D) FISH analysis using an <i>MLL</i> 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 <i>MLL</i> chromosomal translocations. Two hundred cells were analyzed for each sample. Representative images are shown. Red arrows indicate cells positive for an <i>MLL</i> translocation. Panel B shows a cell from subculture 9 in metaphase that was noted to have an <i>MLL</i> translocation.</p

CD34+ cells nucleofected with <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs display enhanced replating potential in semi-solid medium.

<p>Colony forming assays were performed to assess the effect of <i>MLL</i> and <i>AF4</i> or <i>AF9</i> 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 <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs: diffuse colony representing ~80% (left) and compact colony representing ~20% (right) of all colonies.</p

Survival advantage for CD34+ cells containing an <i>MLL-AF4</i> translocation.

<p>Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with <i>MLL</i> and <i>AF4</i> 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 <i>MLL</i> translocations (A) and qPCR for the MLL-AF4 translocation (B). (-- indicates subcultures nucleofected with GFP as negative control)</p

Analysis of dd-ZFNs.

<p>Unless otherwise indicated, rates of gene targeting at day 3 were normalized to the rate of gene targeting achieved using 20 ng of the unmodified ZFNs without drug treatment as this was previously determined to be the conditions used to obtain optimal gene targeting with the unmodified ZFNs. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in <i>HEK</i>293 cells was about 20,000 GFP positive cells per million cells transfected. (A) Titration of transfected DNA of dd-ZFNs in the gene targeting assay with increasing amounts of transfected DNA. (B) Time-course experiment for length of exposure of 1000 nM Shield1 using 5 ng of dd-ZFNs. Hours are given relative to the time of transfection, where “0” is the time of transfection. (C) Drug dose response curve for Shield1 with 5 ng of dd-ZFNs. (D) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in <i>HEK293</i> cells. (E) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in 3T3 cells. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in 3T3 cells was about 20,000 GFP positive cells per million cells transfected (2%). (F) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in HeLa cells per million cells transfected. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in HeLa cells was about 2,000 GFP positive cells per million cells transfected (0.2%). (G) Toxicity assay for all iterations of dd-modified and unmodified ZFNs tested in the gene targeting assay relative to Sce. A value of <100% indicates decreased cell survival as compared with Sce, and demonstrates a toxic effect. Statistical analysis was performed using the Student's T-test comparing ZFN-1/ZFN-2 at 20 ng with no drug treatment to dd-modified ZFNs treated with Shield1. “*” indicates a P-value of <.05 and “n.s.” indicates no statistical significance or a P-value of >.05. Error bars are the standard deviation in measurement of three samples.</p

Characterization of Ub-X-ZFNs that display drug-dependent stability.

<p>(A) Genetic fusion of a ubiquitin moiety to a ZFN with either a “VV” linker or an “R” linker. (B) Expression profile of unmodified and Ub-X-ZFN proteins in the presence and absence of 10 uM of the proteasome inhibitor MG132 from 18–22 hours post-transfection. <i>HEK</i>293FT cells were transiently transfected with vectors encoding either ZFN-1/ZFN-2, Ub-VV-ZFN-1/-2, or UB-R-ZFN-1/ZFN-2. ZFNs were detected using Western blot analysis with an anti-Flag antibody. ZFN-1/ZFN-2 and Ub-R-ZFN-1/ZFN-2 were approximately 37 kD and Ub-VV-ZFN-1/ZFN-2 were approximately 47 kD. The size difference between the Ub-X-ZFNs is due to the Ub-moiety being cleaved off when linked via an R-linker. β-actin serves as a loading control.</p

Characterization of dd-ZFNs that display Shield1-dependent stability.

<p>(A) Genetic fusion of a destabilization domain derived from an FKBP12 mutant to a ZFN. (B) Expression profile of unmodified and dd-ZFN proteins in the presence and absence of 1 uM of the Shield1 from 0–24 hours post-transfection. <i>HEK</i>293FT cells were transiently transfected with vectors encoding either ZFN-1/-2, dd-ZFN-1/-2. ZFNs were detected with an anti-Flag antibody. β-actin serves as a loading control. The molecular weight of the unmodified GFP-ZFNs was approximately 37 kD and for the dd-ZFNs approximately 50 kD.</p