Generation of Ci-Brachyuty-GFP stable transgenic lines in the ascidian Ciona savignyi

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease l-Scel was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for l-Scel. Two founder animals, out of 12 F-0 adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F-2 in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F-3 and F-4 generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos. (C) 2003 Wiley-Liss, Inc.