Construction Of The Transcriptional Coactivator 4 Into The Transgenic Vectors And The Establishment Of Transgenic Technology In Zebrafish

Fish are excellent animal system to be used for gene transfer technology for several reasons. These include large number of embryos produced from one mating, fertilization in vitro, develop of embryos in vitro, easy screening and manipulation of developing embryos under a dissecting microscope. Due to several unique genetic features inherent in zabrafish, such as small size, short lifespan, easy maintenance, and transparent embryos, zebrafish has become a well-established model system and been widely used in gene transfer analyses. Transgenic zebrafish has been applied in the studies of embryonic development of vertebrate, functional genomics, and human genetic diseases.Our previous study indicates that PC4, a universal transcriptional coactivator, plays an important role in early development of vertebrate. In order to establish the platform to further study the functions of PC4 during early development, we intend to set up transgenic technology in fish. As a first step, we constructed wild type PC4 and mutant PC4 genes to pAAV-IRES-hrGFP vector. These constructs were then introduced to zebrefish and bluntsnout bream by microinjection and sperm-mediated gene transfer (SMGT) methods respectively. Unfortunately, we did not get any positive transgenic fish from either of these attempts. A possible explanation could be imputed to the low efficacy of the pAAV-IRES-hrGFP vector to integrate into the genome and the poor efficiency of the home-made device for microinjection. The failure with the sperm-mediated gene transfer method could result from improper voltage used in the electroporation in addition to the inefficient vector.We then switched to pT2AL200R150G as the transgenic vector because it harbors some cis-elements derived from a powerful transposable element, Tol2. When this vector is co-injected with the mRNAs encoding a transposase that specifically recognizes the cis-elements in the vector, the efficiency for the vector to integrate into the fish genome is greatly enhanced. Vector pT2AL200R150G also contains an EGFP gene the protein of which can emit bright green fluorescence when excited by ultraviolet. It is convenient to screen for the transgenic fish by detecting the expression of the EGFP proteins. By co-injecting the pT2AL200R150G vector together with the mRNAs encoding for the transposase into the fertilized eggs from zebrafish, we got 31 EGFP positive embryos from 127 injected embryos. After these EGFP positive embryos have hatched to young fish, only a small number of them continue to express EGFP. This observation suggests that many of the positive embryos seen shortly after injection might represent those in which the transgenes did not eventually integrate into the fish genome.We are in the process of constructing wild type and mutant PC4 genes to pT2AL200R150G+w vector, and using these constructs to produce transgenic zebrafish in order to examine the effects of PC4 on early development. We are also planning to establish the sperm-mediated gene transfer method with these new constructs.