Transgenesis procedures in Xenopus

Abstract

Stable integration of foreign DNA into the frog genome has been the purpose of several studies aimed at generating transgenic animals or producing mutations of endogenous genes. Inserting DNA into a host genome can be achieved in a number of ways. In Xenopus, different strategies have been developed which exhibit specific molecular and technical features. Although several of these technologies were also applied in various model organizms, the attributes of each method have rarely been experimentally compared. Investigators are thus confronted with a difficult choice to discriminate which method would be best suited for their applications. To gain better understanding, a transgenesis workshop was organized by the X-omics consortium. Three procedures were assessed side-by-side, and the results obtained are used to illustrate this review. In addition, a number of reagents and tools have been set up for the purpose of gene expression and functional gene analyses. This not only improves the status of Xenopus as a powerful model for developmental studies, but also renders it suitable for sophisticated genetic approaches. Twenty years after the first reported transgenic Xenopus, we review the state of the art of transgenic research, focusing on the new perspectives in performing genetic studies in this species.

Figure 1. REMI transgenesis procedure

(A) The REMI method has been successfully used to generate transgenic X. laevis tadpoles and lines. Sperm nuclei were isolated, as described by Murray (1991), with the modifications by Kroll and Amaya (1996). The quality and concentration of the sperm solution are determined by Hoechst staining in a haemocytometer (1). The cells are then incubated with the linear transgene along with egg extract and RE. Methods that aimed at improving the REMI method have discarded the use of egg extract and RE (blue box), which made the method very similar to an ICSI protocol (Sparrow et al., 2000). The mixture is then injected into unfertilized eggs (2) and the transplanted embryos are selected at the four-cell stage (3). All eggs are activated by the injection, but only the embryos that cleaved normally are isolated for further analyses. Indeed, as the nuclei are injected at a constant flow rate, at best a third of the eggs are expected to receive a single nucleus. As development proceeds, embryos are scored for the expression of the transgene (4) and placed into an husbandry facility to obtain mature F0 founder animals to derive transgenic lines. Here the generation of an F0 expressing GFP under the control of the ubiquitous CMV (cytomegalovirus) promoter is shown. (B) Tadpole generated using the REMI method expressing a GFP reporter under the control of the cardiac actin promoter. Striated muscle cells of the somites express GFP in an homogenous fashion. (C) Tadpoles expressing a GFP reporter under the control of the cardiac actin promoter generated by microinjection. Microinjected embryos exhibit a punctuated GFP signal in few cells within the somites, reminiscent of a mosaic expression.

Figure 2. Transgenic animals generated by the REMI method: diversity within F0 founders and possible mosaicism

(A) Hemi-transgenic individual showing a left–right restricted distribution of GFP expression using a CMV (cytomegalovirus)–GFP reporter transgene. (B) Hemi-transgenic individual showing an antero–posterior-restricted distribution of GFP expression from a CMV–GFP reporter transgene. (C) Non-mosaic F0 founders showing different level of GFP expression from the same CMV–GFP reporter construct. In all panels, bright-field (BF) images are shown along the GFP pictures. Pictures were taken using the Aequoria system (Hamamatsu).

Figure 3. Publication status on transgenesis in Xenopus

(A) Proportion of PubMed entries taking into account a given model organism and genetically modified animals. The data were obtained by using the following search criteria. For Xenopus, zebrafish, chickens and Caernorhabditis elegans: “Name of the organism”[MeSH] AND Animals, Genetically Modified[MeSH] NOT Mice[MeSH] NOT Drosophila[MeSH]; for mice: Mice[MeSH] AND Animals, Genetically Modified[MeSH] NOT Drosophila[MeSH]; and for Drosophila: Drosophila[MeSH] AND Animals, Genetically Modified[MeSH] NOT Mice[MeSH]. (B) Evolution over years of the number of PubMed entries associated to Xenopus and genetically modified animals. (C) Topic distribution of the PubMed entries referring to both ‘Xenopus’ and ‘Genetically modified animals’. The data were obtained using the topics mentioned on the histogram and the search criteria described above: Xenopus[MeSH] AND Animals, Genetically Modified[MeSH] NOT Mice[MeSH] NOT Drosophila[MeSH]. These searches were performed in October 2007.

Figure 4. Glow in pigmented frogs: the advantage of the albino mutants

GFP fluorescence was analysed in an adult albino frog transgenic for a CMV (cytomegalovirus)–GFP reporter transgene that drives GFP expression ubiquitously. In the left-hand panels, the transgenic albino frog is shown next to a wild-type albino animal, and in the right-hand panels next to a pigmented frog transgenic for the same construct. The pigments obscure the analyses of GFP expression on the dorsal side of the pigmented animal (upper panels). However, when observed on the ventral side, both the albino and pigmented animal express GFP to a similar extent (lower panels). Bright-field images are provided as insets in the main pictures. Pictures were taken using the Aequoria system (Hamamatsu).

Figure 5. Generation of diploid and triploid transgenic lines

(A) Haploid, (B) diploid and (C) triploid individuals can be generated following the transplantation of a random number of male nuclei during the REMI procedure. If the egg does not receive a nucleus, the embryo will be haploid and will not develop beyond the neurula stage (☹), unlike diploid and triploid animals that will give rise to founders (☺). (B) If the egg received a single nucleus, then the embryo generated will produce an heterozygous diploid F0 founder and lines can be derived as shown. Transgenic embryos can be selected by observing the expression of a marker gene, such as GFP, and individuals carrying the transgene (+) are isolated from non-carriers (−). (C) In the case of multiple nuclei transplantation, the embryos generated will be polyploid. It is not known how many nuclei can be maintained in Xenopus. Multiploid cells have been shown to suffer from chromosomal instability and degenerate. However, triploid individuals that result from transplantation of two male nuclei are stable and develop to adulthood (Smith, 1958; Kawahara, 1978; Tompkins, 1978; Tompkins and Reinschmidt, 1991; Noramly et al., 2005). It is not known if these animals are fertile (?) and whether lines can be derived form them. However, triploid embryos could be generated by REMI and maintained in animal facilities. (D) Finally, F0 diploid transgenic female founders can be made homozygous at the F1 generation by using a gynogenesis procedure. In this case, the genomic contribution of the male DNA is eliminated by UV treatment. The UV-treated spermatozoids are used for in vitro fertilization of the transgenic eggs. Inhibition of the first cleavage by pressure re-establishes the diploid state and development can proceed normally.