Studies On Oligomerized Pool Engineering (OPEN) For Zebrafish POP3 And Reporter Assays For POP3 And Its Interaction Protein

Zebrafish has become one of the most important models in vertebrates developmental biology, but the gene knockout method in zebrafish has not yet been well established, which prevents the fully elucidation of biological functions of zebrafish genes. As a result, an international gene knock-out technology was developed in 2008, and this OPEN (Oligomerized pool engineering) is a method of design high affinity zinc-finger nuclease for gene targeting. In this paper, we tried to knockout the POP3 gene in zebrafish using this new method for making a POP3 knockout zebrafish.From the ensembl database(http://www.ensembl.org/), we first abtained the information about the zebrafish POP3 such as the gene location, exons, introns and ORF. The potential full ZFN sites that were best suitful for POP3 coding sequence was obtained by the zinc-finger targeter (ZiFiT) program for the following study. For each half binding site, three single ZFPs were then amplified from ZFN libraries, and linked them together with those ZFP fragments amplifed by PCR. The combinatorial zinc-finger library was transferred into E. coli cells. Using OPEN method, six three-finger arrays for each half site were selected, and the activation of those ZFPs were analzed by using bacteria double hybrid (B2H). As a result, we successfully obtained a pair of ZFPs that bind to the 355 bp site, which will be used for the POP3 targeting zebrafish.The results obtained by our lab showed that ATF4 can interact with POP3. We used gene reporters to study the interaction between POP3 and ATF4 and proved that POP3 and ATF4 can up-regulate the expression of GATA4 in the heart development. An analysis of GATA4 promoter sequence by using Bioinformatics method showed that there is a conservative CRE site in 2000 bp upstream of human GATA4 transcription starting sites. We cloned the human GATA4 promoter sequence that was transferred into a reporter plasmid. By analysis of luciferase assay system, it was showed that the CRE binding site is critical for POP3 and ATF4. Based on the results above obtained by reporter assays, we can conclude that POP3 interacts with ATF4, and both synergistically regulate the expression of GATA4 through binding to the CRE site in the GATA4 promoter.