Knockout HSF1 In Zebrfafish By TALEN Technology

Heat shock transcription factors?HSFs?, also named as heat shock factors, which are a group of ubiquitous expressed eukaryotic transcriptional factors with highly homology in structure and function. When cell suffers from heat shock or other harsh stimulus, HSFs transactivates a doses of heat shock proteins?HSPs? to help cells to recover and survive. HSFs family includes four members. HSF1 is the major one responsible for the expression of HSPs and conserved in mammals. Recent studies have shown that HSF1 is involved the control of many key physiological processes such as growth and development, cell apoptosis and differentiation, inflammation response and carcinogenesis.HSF1 plays a pivotal role in the lens development. It is has been reported that in the lens epithelial cells of HSF1 knockout mice, it binds competitively to promoters of HSF4's target genes and regulate the lens development. What's more, as indicted by the HSF1 knockdown zebrafish, it may regulate the lens development by activation the expression of HSP70. Because its embryo is transparent and can be observed since it hatch, zebrafish is more suitable to study the early development of eye than mice. However, no HSF1 genetic knockout model of zebrafish has been reported, it is necessary to build it.TALEN?Transcription activator-like effector nuclease? is a recent developed technology for genome editing. It takes advantage the site specific TALE?transcription activator-like effector? modular to bind the flanking sequence of the TALEN targets and employs the non-specific Fok I endonuclease to cleave the target sequence, which results in DNA double-strand breaks and induces NHEJ DNA repair and then introduces mutations within the target sequence with a high probability.In this thesis, we first determined the spatial-temporal expression pattern of zebrafish HSF1 by semi-quantitative RT-PCR. In the embryonic stage, the expression of HSF1 reached the peak at 6 hpf?hours post fertilization? and down to the bottom at 48 hpf. Although lower than the early embryonic stage, the expression of HSF1 is increased at 72 hpf, which indicates its maternal expression. In the adult tissues, HSF1 is ubiquitously expressed and has the highest level in the eye, ovary and testis expression, and lower level in the heart, spleen and other tissues. These evidences suggest HSF1 plays important physiological roles in various organs, especially the eye, ovary and testis.Next, we built a HSF1 knockout zebrafish model by the TALEN technology. The TALEN target sequences of zebrafish HSF1 was located in its exon 1 and designed by the online software, then we constructed the TALEN expression plasmid by the Golden Gate kit. After linearization and in vitro transcription, the synthesized TALEN mRNA was injected into the fertilized zebrafish embryo at one cell stage. After checking the TALEN efficiency by PCR-RFLP, we obtained the F0 chimeric mutant. By crossing the F0 and wild type zebrafish, we successfully got three types of F1 zebrafish, namely: c.90₉8delGGATCCGGA?p.Glu30_Pro32del?, c.78₈1delGACG?p.Trp26 Cysfs * 10? and c.91delG?p.Asp31 Ilefs * 6?.In this study, we constructed the hsf1 gene knockout zebrafish by TALEN technology for the first time, and we obtained three types of HSF1 defect zebrafish finally. This work laid solid foundations for the functional study of HSF1, especially its molecular role in the zebrafish eye development in the future.