Fish MB et al. (2012), Simple, fast, tissue-specific bacterial artific...

XB-ART-44331

Genesis 2012 Mar 01;503:307-15. doi: 10.1002/dvg.20819.

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Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.

Fish MB , Nakayama T , Grainger RM .

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We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.

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Species referenced: Xenopus tropicalis
Genes referenced: atoh7 pax6 rax

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	Figure 1. Expression of the pax6 dual reporter BAC in Xenopus embryos. (a) Detail of the pax6 coding region contained in BAC 109E08, showing construction of the pax6 BAC dual reporter injected into Xenopus tropicalis embryos shown in f, r, and t, and into the X. laevis embryo shown in q. Exons are numbered and shown as grey boxes. Alternate translational start sites indicated with TG,with the most common isoforms using the exon 4 start site, and the shorter, retinal specific isoform using exon 8 start site. (b) In situ hybridization of uninjected siblings using a probe against pax6 mRNA to show endogenous expression (b, stage 18, anterior view; c, stage 23; d, stage 33; e stage 47 dissected gut). Expression is detected in the developing eye, brain, pancreas, and intestine; developing eye indicated by arrows in c and d, pancreas indicated by arrow in e, intestinal expression indicated by arrowhead in e. (f) In situ hybridization with probe detecting gfp3 mRNA in BAC-injected embryos (f, stage 18, anterior view; g, stage 23; h, stage 33; i stage 47 dissected gut). Expression of the transgene is detected in the same tissues as endogenous pax6 expression: developing eye, brain, intestine, and pancreas; pancreas indicated by arrow in i, intestinal staining indicated by arrowhead in i. (The dark coloration of intestines is due to pigment remaining in tissues.) (j) Fluorescent images of gfp3 expression at same stages as in situ hybridized embryos above. Developing eye indicated by arrows in k and l. (m) Detail of stage 47 embryo gut. gfp3 speckling is seen in the intestine, indicated with arrowhead. Although pancreatic expression of BAC gfp3 mRNA is confirmed via in situ hybridization for gfp3, gfp3 protein is not observed in the pancreas. (n) Detail of retina and lens of stage 47 embryo injected with dual reporter. The more common isoforms of pax6 reported by the gfp3 transgene are seen widely expressed in both the lens and retina, while the less expressed, shorter isoform reported with the mkate2 transgene is excluded from the lens, and detected in only a subset of retinal cells. (q) gfp3 expression seen in a stage 33 X. laevis embryo also recapitulates endogenous expression. (r) gfp3 expression in a stage 47 embryo injected with the pax6 reporter BAC. Expression is still detected in the retina and lens, olfactory bulbs (arrows) and optic nerves (arrowheads). (s) Comparison of stage 33 embryos, dorsal view of head: pax6 endogenous expression (s), gfp3 transient expression from pax6 BAC (t), and gfp3 expression seen in an F1 embryo from an integrated line in which gfp3 expression is driven by the upstream 3.6 kb pax6 promoter region (u). (j) Embryos were PTU-treated to visualize retinal tissue. l = lens, r = retina.

References [+] :

Andreazzoli, Xrx1 controls proliferation and neurogenesis in Xenopus anterior neural plate. 2003, Pubmed, Xenbase