Enhancer Activity Detection and Mutation Analysis

The ETS1 overexpression construct generated from the ETS1 cDNA sequence and the pcDNA3.1 vector was kindly provided by Professor G. Huang from the Children’s Hospital of Fudan University. The cDNA sequences of NKX2.5 and JUN, which were cloned into the pENTER vector (Vigenebio, China), were synthesized by Shanghai Sunny Biotechnology Co., Ltd. The zebrafish and human DNA sequence of the enhancer was downloaded from the ECR Browser. DNA regions of the candidate enhancers were amplified by PCR, cut with XhoI and BglII and cloned into pCNE7.04-E1b-GFP-T2KXIGQ, the enhancer activity detection vector (Li et al., 2010 (link)) (Fig. 1A). To construct a luciferase reporter plasmid (Fig. 1B), enhancer fragments were cut with KpnI and XhoI, and the original SV40 promoter of the luciferase reporter vector pGL3-promoter (Promega; USA) was replaced by E1b, which is a widely used basic promoter. We termed this new vector pGL3-E1b. In the mutated enhancer constructs, a single base in the TFBSs was mutated but was not introduced a new heart-related TFBS. The primer sequences used for PCR amplification of the enhancer activity detection and mutation analysis constructs are listed in Table 1.