pTransgenesis: a cross-species, modular transgenesis resource.
As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.
PubMed ID: 22110059
PMC ID: PMC3222217
Article link: Development.
Grant support: Wellcome Trust
Genes referenced: cfp lgals4 pvrl2 tubb2b vim
Article Images: [+] show captions
|Fig. 2. Incorporation of Xenopus-compatible elements into pTransgenesis. (A) Schematic of recombination with a Xenopus-compatible p4 vector containing a single I-SceI site, Tol2 elements, and SAR-CH4 sequences, p3 Katushka RFP and Xenopus-compatible p1 and p2 constructions. (B-K) Transgenic tadpoles from the resulting recombinations are shown. Individual p1, p2, p3 constructions and expression domains are written on each panel in white. Images in E′ and G′ are magnified images of the boxed regions in E and G, respectively. Image in F′ shows the induction of RFP from the heat-shocked tadpole in F.|
|Fig. 3. A highly efficient method of testing promoters and creating transgenic lines using pTransgenesis. (A) Genomic PCR fragments are cloned directly into the p2 position, thus allowing the recombination shown. (B,C) PCR products encoding regions 5′ to nectin-2 and vimentin were generated and tested for transcriptional activity in F0 X. laevis and in F1 X. tropicalis. (D) Transverse sections through the hindbrain and spinal cord of embryos (dorsal side up) stained for endogenous vimentin expression (upper panels), or Venus transgene expression (green) in F0 transgenic X. laevis (middle panels) and F1 transgenic X. tropicalis (lower panels). (E) Transverse sections through the neural plate of stage 18 embryos (dorsal side up) stained for endogenous nectin-2 expression (upper panel), or Venus transgene expression (green) in F0 transgenic X. laevis (middle panel) and F1 transgenic X. tropicalis (lower panel). Nuclei are stained with DAPI (blue) in middle and lower panels in D and E.|
|Fig. 4. pTransgenesis in various models. (A) Schematic showing plasmid recombinations yielding plasmids compatible with transgenesis in HeLa cells, Xenopus, zebrafish and Drosophila. (B) Images of zebrafish injected with the indicated p1, p2, p3 and p4 elements with or without Tol2 mRNA. Open arrowhead points to activity of the p1 γ-crystallin RFP marker. (C,D) Results from HeLa cell transfections and puromycin (Puro) selection using pTransgenesis-engineered constructions. Red open arrow indicates dying cells. (E) Drosophila melanogaster engineered with the indicated pTransgenesis constructs versus wild type. (F,G) Phase contrast and fluorescence image of pTransgenesis-engineered Drosophila embryo. Open and closed arrowheads indicate gut and cuticle autofluorescence, respectively. (H-J) Confocal images from the indicated Gal4 crosses. (H) srp-Gal4, stage 16 embryo, GFP-labelled hemocytes. (I) eval-Gal4, stage 16 embryo, GFP-labelled CNS. (J) engrailed-Gal4, ventral view of stage15 embryo during dorsal closure process, GFP-labelled engrailed domain segments. Scale bars: 25 μm in C,D; 100 μm in F-J.|
|Fig. S1. Germline transmission of pTransgenesis constructs using I-SceI. (A) The construction shown was used to create a transgenic line in X. laevis using I-SceI-mediated transgenesis. The p2 NBT element drives RFP expression in differentiated neurons. (B) Fluorescent images derived from the F1 generation of this transgenic line. (C) Enlarged view of white box in B showing optic nerve (on) and olfactory placode (op).|
|Fig. S2. A p3 library of fluorescent proteins. (A) Fifteen fluorescent proteins with the approximate peak emissions indicated were incorporated into the p3 coding sequence position of pTransgenesis. Note: Kaede* and Kikume* possess red-shifted emissions following photoactivation. (B) The p3 fluorescent proteins were linked downstream of the p2 CMV promoter and with a p1 γ-crystallin selection marker. (C) Resulting constructs were tested for functionality via REMI. Note: fluorescence was captured using CFP, GFP or RFP filters and then pseudocoloured to represent each protein's peak emission more accurately. The Kaede protein has undergone a red-green colour shift after exposure to 405 nm light.|
|Fig. S3. Establishment of X. laevis transgenic lines amenable to Gal4-UAS binary transgene expression. (A,B) Schematics showing the construction of Gal4 and UAS constructs using pTransgenesis plasmids. (C-E) Merged brightfield and fluorescent exposures of single transgenic embryos containing the indicated Gal4 or UAS drivers. (F-H) Merged brightfield and fluorescent exposures of double transgenics. The arrowheads in G and H show a morphological defect in the tail in severe and less severe phenotypes. (I) Schematic of F0 founder crossings. (J) Brightfield images showing resulting F1 transgenic tadpoles, with double transgenics showing germline transmission of tail and body length defect. (K) Merged fluorescence of GFP and RFP channels for the tadpoles shown in I.|
|Fig. S4. Establishment of an X. tropicalis transgenic line amenable to Cre-loxP fate mapping. (A) Schematic of the pTransgenesis construct used to generate an X. tropicalis line via I-SceI transgenesis. (B-E) Brightfield (B,C) and fluorescence (D,E) imaging of the F1 generation. Note that GFP expression is visible in only one eye lens in D and E, as the other lens is hidden behind the pigmented epithelium of the other eye. (F) Schematic of experiment to determine induce Cre-mediated site-specific recombination, which places mCherry under the control of the CMV promoter, and thus the functionality of the loxP elements in p3. (G-N) Hemispheric expression of cre mRNA in F2 generation results in Cre-mediated expression of mCherry, and concomitant loss of eCFP expression in the progeny of the cell that was injected with cre mRNA at the one cell stage (K-N) compared with control uninjected embryos (G-J).|