Evaluation of CERT gene disruption by TALEN-CERT pairs in HeLa cells.

A, Target sites of TALENs-CERT pairs (version 1 and 2) in human CERT gene. The sequences are located in exon 2, which codes part of the PH domain. The target sites are shown in bold. The numbers on the right and left of the sequence indicate the sequence numbers from the A of the translation initiation codon, based on CERT mRNA (accession number AY4533859). B, Resistance to lysenin in TALEN-CERT–treated HeLa cells. Six TALEN-CERT pairs, which contain two pairs of repeat modules directed against different target sites (CERTv1 and v2) contained in three TALEN scaffolds (TAL original (ori), TAL-ModA, and TAL-ModB), as well as the empty vector (–). These TALEN pairs were transfected into HeLa-mCAT#8 cells, followed by 30°C incubation. The cells were treated with lysenin at 800 ng/ml for 2 h. Their viability was estimated by the MTT assay and is expressing as a percentage of the value (OD570) in the absence of lysenin: mean percentage ± S.D. obtained from three independently repeated experiments. The Bonferroni corrected t-test was used for multiple comparisons. *, pA, TALEN（转录激活因子样效应物核酸酶，Transcription Activator-Like Effector Nucleases）-CERT对（版本1与版本2）在人CERT基因中的靶位点。所述序列定位于外显子2，该外显子编码PH结构域（Pleckstrin Homology domain）的部分序列。靶位点以粗体显示。序列左右两侧的数字代表基于CERT mRNA（登录号AY4533859）的翻译起始密码子A的序列编号。B, 经TALEN-CERT处理的HeLa细胞对溶血素的抗性。本次实验共设置6组TALEN-CERT对，其中包含两组靶向不同靶位点（CERTv1与v2）的重复模块，分别搭载于3种TALEN骨架（原始TAL（ori）、TAL-ModA及TAL-ModB），并设置空载体对照组（–）。将上述TALEN对转染至HeLa-mCAT#8细胞后，于30℃孵育。随后以800 ng/ml浓度的溶血素处理细胞2小时，采用MTT法（MTT assay）检测细胞活力，并以未添加溶血素组的OD570值为基准，将细胞活力表示为其百分比：数据为三次独立重复实验所得的平均值±标准差（S.D.）。多重比较采用Bonferroni校正t检验，*，p