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Keywords:

  • ascidian;
  • transgenesis;
  • microinjection;
  • I-SceI;
  • notochord morphogenesis;
  • mutant

Abstract

Summary: 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 I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F0 adults tested, were found to transmit the transgene to their offspring (F1s) at frequencies of 42% and 23%. The transgene was further inherited by the F2 in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F3 and F4 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. genesis 35:248–259, 2003. © 2003 Wiley-Liss, Inc.