XB-ART-39882Dev Dyn. July 1, 2009; 238 (7): 1727-43.
Transgenesis in Xenopus using the Sleeping Beauty transposon system.
Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon. SB has been used extensively in human cell lines and in whole animal vertebrate model systems such as the mouse, rat, and zebrafish. In this study, we describe the use of SB in the diploid frog Xenopus tropicalis to generate stable transgenic lines. SB transposon transgenes integrate into the X. tropicalis genome by a noncanonical process and are passed through the germline. We compare the activity of SB in this model organism with that of Tol2, a hAT (hobo, Ac1, TAM)-like transposon system.
PubMed ID: 19517568
PMC ID: PMC2848081
Article link: Dev Dyn.
Grant support: 5R25CA023944 NCI NIH HHS , R01 HD042294 NICHD NIH HHS , R01 HD042294-01 NICHD NIH HHS , R01 HD042294-01S1 NICHD NIH HHS , R01 HD042294-02 NICHD NIH HHS , R01 HD042294-03 NICHD NIH HHS , R01 HD042294-04 NICHD NIH HHS , R01 HD042294-05 NICHD NIH HHS , R01 MH079381-01A2 NIMH NIH HHS, R01 MH079381 NIMH NIH HHS, R25 CA023944 NCI NIH HHS
Genes referenced: actl6a kidins220 myh3 tcim
Article Images: [+] show captions
|Fig. 3. Southern blot analysis of genomic DNA harvested from SB-mediated transgenic Xenopus tropicalis and Xenopus laevis. Genomic DNA harvested from progeny of each of the founder animals was digested with BglII and separated by electrophoresis on an agarose gel, transferred to a membrane and probed with a radiolabeled green fluorescent protein (GFP) -encoding DNA fragment. a: Schematic representation of the pT2GFP SB transposon indicating the approximate position of the unique BglII site and region used for the probe (bar). Not to scale. b: Southern blot analysis of pT2GFP transgenic founders. Outcross of founder 4M resulted in two discrete hybridization patterns indicating independent segregation of the alleles (compare samples 4M-1 and 4M-2). The founder animal contains, at least, four copies of the GFP sequence that are inherited by the progeny (open and closed triangles). Likewise, the progeny of 8F display two different hybridization patterns (compare 8F-1 and 8F-3). Founder 8F also contains at least four copies of the transgene (carets [!] and asterisks [*]). Progeny from founder 7M have a complex hybridization pattern suggesting the presence of a concatamer of transposon transgenes. Tadpoles 4M-1 and 7M have hybridizing bands (labeled open triangle and #) that migrate faster than the predicted lower limit for the BglII digested transgene (2.97 kb; see Fig. 1a). This indicates that the integration events at these loci are complex and have involved fragmentation of the transposon transgene. Size markers (in kb) are indicated on the left side of the blot. c: Enlarged view of the Southern blot to illustrate that founder 6M has two closely migrating bands (*).d: Southern blot analysis of Xenopus laevis founder lines L2M, L3M and L6M. Genomic DNA samples for three GFP-positive F1 animals (#1, 2, and 3) and a GFP-negative F1 sibling (#4) from each founder line were digested with BglII, separated on a 0.7% (w/v) agarose gel, and probed with a 700-bp GFP fragment as in Figure 3b. L2M founder line has at least five hybridizing GFP bands, L3M has at least three GFP-positive bands, and L6M has two GFP-positive bands.|
|Fig. 4. Fluorescence in situ hybidization (FISH) of cells harvested from pT2GFP transgenic Xenopus tropicalis. Interphase nuclei were prepared from circulating blood cells harvested from individual tadpoles and probed with fluorescein isothiocyanate (FITC) -labeled green fluorescent protein (GFP) for detection. White arrows indicate location of the GFP probe in the samples. a: pT2GFP X. tropicalis founder line 4M. b: pT2GFP founder line 5M. c: pT2GFP founder line 6M. d: pT2GFP founder line 7M. e: Interphase nuclei and metaphase spread of founder line 7M.|
|Fig 7. Southern blot analysis of F1 tadpoles from SB11-mediated pT2GFP transgenic Xenopus tropicalis founders. Founder male 622E has two independently segregating insertion events; compare female 622E1 (four green fluorescent protein [GFP] -positive hybridizing bands) and 622E3 (three GFP-positive hybridizing bands). Tadpole female 622E2 has inherited both integration events. F1 progeny from founders male 623F, male 2262, and male 2232 have two GFP-positive hybridizing bands.|