Cardiacspecific Transgenic Lines

The current limitation on establishment of cardiac-specific transgenic reporter lines reflects the dearth of well-characterized promoters in Xenopus, rather than difficulties inherent in the transgenesis technique. From our own studies, we have identified regions of the Nkx2-5, cardiac actin, and myosin light-chain 2a (MLC2a) promoters that confer the appropriate patterns of transgene expression, and we are using these to construct transgenic GFP reporter lines in Xenopus laevis and Xenopus tropicalis. Of these, the best characterized is the X. laevis cardiac actin line which utilizes 570 bp of promoter sequence and is currently bred to the F2 generation (Latinkic et al. 2002). Like its endogenous counterpart, the GFP reporter is expressed in both skeletal and cardiac muscle of embryos from the onset of differentiation (Fig. 1). Interestingly, little or no expression is detected in adult frogs, indicating that additional regulatory elements are necessary to maintain expression after metamorphosis. The MLC2a transgene is expressed exclusively in the myocardial layer of the developing heart, whereas the XNkx2-5.GFP transgene is expressed within the cardiac mesoderm prior to the onset of terminal differentiation and also within the underlying endoderm (Sparrow et al. 2000b). No promoters are yet characterized from Xenopus that can be used to drive expression specifically within the endocardium, al

Figure 1. Expression of a cardiac actin.GFP transgene. A 570-bp portion of the proximal promoter containing conserved CArG box and E box motifs (diagram) is first expressed in the differentiating skeletal muscle of the myotomes in the neurula embryo (A). In the feeding tadpole (B, ventral view), expression is now also evident in musculature of the pharynx and in the heart. (ot) Outflow tract; (V) ventricle.

Figure 1. Expression of a cardiac actin.GFP transgene. A 570-bp portion of the proximal promoter containing conserved CArG box and E box motifs (diagram) is first expressed in the differentiating skeletal muscle of the myotomes in the neurula embryo (A). In the feeding tadpole (B, ventral view), expression is now also evident in musculature of the pharynx and in the heart. (ot) Outflow tract; (V) ventricle.

though this might be possible using a portion of the SMAD 3 promoter (Howell et al. 2001). Endothelial markers might provide an alternative, and the Fli.GFP reporter used so successfully to map development of the vasculature in zebrafish embryos (Lawson and Weinstein 2002) also shows similar expression in tadpoles (T. Mo-hun, unpubl.). From studies in chick and the mouse, we might expect that the IRX4 and ANF promoters would provide a means of targeting transgene expression to the ventricular and atrial myocardium, respectively (Bao et al. 1999; Christoffels et al. 2000). However, in Xenopus, both genes are actually expressed throughout the myocardium during the period of heart chamber formation (Small and Krieg 2000; Garriock et al. 2001) and, at least in the case of the ANF promoter, atrial restriction is only established later.

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