Cryopreservation of Xenopus transgenic lines

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Abstract
Xenopus laevis has been widely used for molecular, cellular, and developmental studies. With the development of the sperm‐mediated transgenic method, it is now possible to study gene function during vertebrate development by using this popular model. On the other hand, like other animal species, it is labor intensive, and the maintenance of transgenic lines is expensive. In this article, we investigated the possibility of using sperm‐cryopreservation as a means to preserve transgenic frog lines. We demonstrated that cryopreserved sperms are viable but not fertile under our in vitro fertilization (IVF) conditions. However, by microinjecting cryopreserved sperm nuclei, we successfully regenerated a transgenic line carrying a double promoter transgene construct, where the marker gene encoding the green fluorescent protein (GFP) is driven by the γ‐crystallin gene promoter and a gene of interest, encoding a fusion protein of GFP with the matrix metalloproteinase stromelysin‐3 (ST3‐GFP), is driven by a heat shock‐inducible promoter. We demonstrated the functional transmission of the ST3‐GFP transgene by analyzing the phenotype of the F1 animals after heat‐shock to induce its expression. Our method thus provides an inexpensive means to preserve transgenic frog lines and a convenient way for distribution of transgenic lines. Furthermore, the ease with which to microinject nuclei compared to the technically demanding transgenesis procedure with variable outcome should facilitate more laboratories to use transgenic Xenopus laevis for functional studies in vivo. Mol. Reprod. Dev. 67: 65–69, 2004.