Xenopus laevis has been widely used for molecular, cellular, and developmental studies. With development of the sperm-mediated transgenic method, it is now possible to study the gene function during vertebrate development by using this popular model. On the other hand, maintenance of transgenic LINES is both expensive and labor-intensive, as for other animal species. In this study, we investigated the possibility of using sperm cryopreservation as a means of preserving the transgenic frog LINES.
We demonstrated that cryopreserved sperms are viable but not fertilizing under in vitro fertilization conditions. However, by microinjecting the nuclei from cryopreserved sperms, we could successfully regenerate the transgenic line carrying a double promoter transgene construct. One
promoter drove the marker gene encoding the green fluorescent protein (GFP), and the second, the heat shock-inducible promoter, drove the expression of GFP fused to matrix metalloproteinase stromelysin-3 (ST3-GFP). We demonstrated the functional transmission of the ST3-GFP transgene by analyzing the phenotype of the F1 animals after heat-shock induction of the transgene expression.
Our method thus provides an inexpensive means to preserve transgenic frog LINES and a convenient way for distributing the transgenic LINES for studies. Furthermore, easy microinjection of nuclei compared to the technically demanding transgenesis procedure with variable outcomes can encourage more laboratories to use transgenic Xenopus laevis for functional studies in vivo.