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.
Image published in: Yergeau DA et al. (2009)
Copyright © 2009. Image reproduced with permission of the Publisher, John Wiley & Sons.
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