XB-ART-44331Genesis March 1, 2012; 50 (3): 307-15.
Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.
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.
PubMed ID: 22084035
PMC ID: PMC3870158
Article link: Genesis
Grant support: EY017400 NEI NIH HHS , EY019000 NEI NIH HHS , R01 EY017400 NEI NIH HHS , R01 EY018000 NEI NIH HHS , R43 EY019000 NEI NIH HHS , R44 EY019000 NEI NIH HHS
Genes referenced: atoh7 pax6 rax
Article Images: [+] show captions
|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.|
|Figure 2. Expression of the rax-gfp3 fusion BAC. (a) Detail of rax coding region contained in BAC 349A23, showing construction of the rax-gfp3 fusion construct. (b) Bright field view of stage 24 embryo injected with rax-gfp3 fusion BAC. (c) GFP fluorescent view of same embryo, showing expression of transgene in developing retina. (d) In situ hybridization against gfp3 mRNA in BAC-injected, stage 24 embryo. (e) In situ hybridization of WT sibling embryo showing endogenous rax mRNA expression. (f) rax overexpression phenotypes seen in stage 42-43 tadpoles injected with rax-gfp fusion BAC. Arrows indicate formation of ectopic retinal pigmented epithelium. Arrowheads indicate eyes shifted medially. (i) rax overexpression phenotypes observed in rax mRNA injected embryos. Arrows and arrowheads indicate the same structures as in f. (j) Normal, wild-type stage 42 embryo for comparison. Reproduced with permission of the Publisher, John Wiley & Sons.|
|Figure 3. Expression of the xath5 GFP reporter BAC. (a) Detail of xath5 coding region contained in BAC 38N10, showing replacement of coding region with gfp3 open reading frame (ORF). (b) Bright field image of stage 35 embryo injected with xath5 reporter BAC. (c) gfp fluorescence image of same embryo, showing gfp3 expression in developing retina. (d) In situ hybridization against gfp3 mRNA in BAC-injected, stage 35 embryo. (e) In situ hybridization of WT sibling embryo showing endogenous xath5 mRNA expression. (f) Stage 46 F2 tadpole from integrated line, using xath5 promoter to drive GFP expression. (g) Stage 46 xath5 BAC-injected tadpole GFP expression is comparable to integrated line expression. Embryos were PTU treated to visualize retinal tissue (b). Reproduced with permission of the Publisher, John Wiley & Sons|
|Figure 4. BAC constructs replicate more efficiently in developing embryos than smaller plasmids. (a) Schematic showing pax6 dual reporter BAC injected into embryos (40 pg). DNA gel image shows results from PCR assay amplifying gfp3 at the indicated stages. (b) Schematic showing plasmid reporter injected into embryos for PCR replication assay. DNA gel images show PCR results from two different DNA amounts injected (40 pg and 4 pg, as indicated). (c) pax6 promoter-GFP in BAC vector construct injected into embryos for PCR assay. Gel shows PCR results from embryos collected at indicated stages. (d) Graph shows results of quantification of gel bands from three independent experiments performed for each construct, and though these experiments were done independently using slightly different PCR conditions, they are shown together in Figure 4d to illustrate the difference in patterns in the two experiments. Error bars show standard deviations, and asterisks indicate P values <0.05 for transgene amplification as compared to 1 hpf at stages 9 (P = 0.016), 25 (P = 0.037), and 35 (P = 0.016). (e) Consistent expression of transgene is observed in pax6 BAC injected embryos. (f) Ectopic, mosaic, and inconsistent expression observed in plasmid injected embryos. Reproduced with permission of the Publisher, John Wiley & Sons|