Construction of an expression vector containing a ?-actin promoter region for gene transfer by microinjection in red sea bream Pagrus major

Abstract

Transgenic technology has been widely applied to a variety of freshwater fish species. However there are few reports on the use of this technology in commercially important marine species. In this study, the construction of expression vectors containing the b-actin promoter region for use in the red sea bream Pagrus major, a species of considerable importance to the aquaculture industry in Japan is reported. The b-actin gene was cloned from a red sea bream genomic DNA library. Recombinant plasmids were constructed by linking the 5′ flanking region of the b-actin gene to the green fluorescent protein reporter gene, followed by the poly A signal sequence of simian virus 40 or the 3′ flanking region the b-actin gene. Expression of these constructs was examined following microinjection into zebrafish and red sea bream embryos, and compared to that of the expression vector pXI-GFP driven by the Xenopus elongation factor 1a. The results indicated that the construct consisting of the b-actin 5′-and 3′ flanking regions was the most efficacious. In future studies, it is planned to investigate the efficient condition for integration into chromosomes of the transgene.

Generation of transgenic medaka expressing claudin7-EGFP for imaging of tight junctions in living medaka embryos

“…We then constructed a vector that expresses cld-7-EGFP under the influence of the red seabream β-actin promoter (Kato et al 2007) to generate a transgenic medaka that stably expressed the fusion protein ubiquitously (Fig. 2b).…”

“…The claudin-7-EGFP mRNA was synthesized in vitro by using a Message Machine kit (Ambion, Austin, Tex.). The claudin-7-EGFP fragment was subcloned downstream of the red seabream β-actin promoter (Kato et al 2007) Whole-mount in situ hybridization Embryos were dechorionated by using proteinase K and the hatching enzyme of medaka, fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), and stored in methanol at -20°C. After dehydration, embryos were treated with proteinase K and refixed with 4% PFA/PBS.…”

Perkembangan Rekayasa Genetika Dalam Budidaya Ikan Hias Di Indonesia

“…Metode transfer gen yang bertujuan untuk pengujian aktivitas promoter salah satunya dengan metode transfeksi ke sel kultur (Kato et al, 2007). Pada metode ini yang diperlakukan adalah telur yang baru keluar dan sebelum terjadi pembelahan 1 sel.…”

Aktivitas Promoter â-aktin Ikan Medaka Jepang (Oryzias latipes) pada Ikan Mas (Cyprinus carpio)

“…Pengujian aktivitas promoter umumnya dilakukan dengan cara menginjeksikan konstruksi gen ke embrio dan kemudian mengamati ekspresi sementara (transient) dari gen penanda yang digunakan (Maclean et a, 2002;Takagi et al, 1994;Tsai et al, 1995;Maclean et al, 1996;Muller et al, 1997;Hamada et al, 1998;Alimuddin 2003;Kato et al, 2007) atau membuat ikan transgenik (Higashijima et al, 1997). Metode lain yang juga bisa digunakan adalah menginjeksi langsung konstruksi gen ke otot daging (Hansen et al, 1991;Rahman & Maclean 1992) atau transfeksi ke sel kultur (Hwang et al, 2003;Kato et al, 2007 ). Menurut Etkin et al, (1984 cit Winkler et al, (1991)…”