Site-specific transgenesis in Xenopus

Abstract

Transgenesis is an essential, powerful tool for investigating gene function and the activities of enhancers, promoters, and transcription factors in the chromatin environment. In Xenopus, current methods generate germ-line transgenics by random insertion, often resulting in mosaicism, position-dependent variations in expression, and lab-to-lab differences in efficiency. We have developed and tested a Xenopus FLP-FRT recombinase-mediated transgenesis (X-FRMT) method. We demonstrate transgenesis of Xenopus laevis by FLP-catalyzed recombination of donor plasmid cassettes into F(1) tadpoles with host cassette transgenes. X-FRMT provides a new method for generating transgenic Xenopus. Once Xenopus lines harboring single host cassettes are generated, X-FRMT should allow for the targeting of transgenes to well-characterized integration site(s), requiring no more special reagents or training than that already common to most Xenopus labs.

Figure 1. Generation of F₀ FRMT frogs with host cassette integration site(s)

Restriction-enzyme-mediated integration (REMI) was used to introduce the acceptor plasmid pf1CarAct→eGFPf3 (A) containing FRT1 and FRT3 sites flanking the Xenopus laevis cardiac actin promoter driving eGFP expression. The locations of primers used to identify transgenic animals are indicated. (B) PCR amplification from genomic DNA isolated from web clippings of F₀ FRMT frogs. H4 primers served as a loading control. The percentage of eGFP fluorescent F₁ animals generated by either natural matings of FRMT male (♂) with wild-type females or in vitro fertilization of eggs from FRMT females (♀) with wild-type sperm is indicated. The number of uninjected tadpoles scored is also shown. Male E failed to fertilize any eggs in three natural mating attempts. Female G had developmental defects and was therefore not induced to lay eggs. Females D and J were used to test for FRMT activity. (C) Brightfield and fluorescent image of stage 24 F₁ embryos generated by in vitro fertilization of F₀ female D eggs with wild-type sperm. P: plasmid, WT: genomic DNA from wild-type control, ND: not determined. Scale bar: 1 mm.

Figure 2. Design of the Xenopus FLP Recombinase Mediated Transgenesis (X-FRMT) system

(A) A donor vector (pf1MCSpolyAf3) containing a multiple cloning site (MCS) followed by the SV40 polyadenylation sequence was generated. The MCSpolyA was flanked by FRT1 and FRT3 sites. Promoters/enhancers (in this example the Xenopus laevis −508→+41 rhodopsin promoter) and marker sequences (in this case mCherry fluorescent protein) are cloned into the MCS to generate donor constructs. Injection of the donor construct and complementary RNA (cRNA) coding for the Flippase recombination enzyme (FLP) into F₁ embryos from F₀ FRMT frogs results in site-specific integration of the transgene into the host genome. Resulting transgenic embryos may express eGFP-only (no recombination), mCherry-only (complete recombination), or both. Primer pairs specific to the host genome (eGFP) and donor construct (mCherry) can confirm X-FRMT. (B) FLP binds to the 13-bp R2 and inverted repeat (IR1) sequences, cleaving the top and bottom DNA strands immediately before and after the 8-bp asymmetric core region, respectively. Recombination only takes place between identical FRT sites (R2-Core-IR1), allowing directional recombination in the system.

Figure 3. Recombinase-mediated cassette exchange of the XOP→mCherry for the CarAct→eGFP transgene

D♀ eggs were in vitro fertilized with wild-type sperm and injected with donor DNA only (pf1XOP→mCherryf3) or with FLP cRNA. (A) Eight weeks post-fertilization the percent of eGFP-positive and mCherry-positive embryos were determined. (B) Seven of eleven (64%) uninjected embryos tested, were genotyped PCR-positive using eGFP-specific primers. (C) Eleven of twelve (92%) embryos selected as eGFP-negative and fluorescent for mCherry in the eyes were genotyped PCR-positive using mCherry-specific primers (Note: very faint products were detected in F₁ tadpoles 11 & 12). None of the mCherry-positive animals were found to contain the CarAct→eGFP transgene. C: control (no template); U1 & U2: gDNA from uninjected eGFP-positive and eGFP-negative F₁ tadpoles, respectively.

Figure 4. Cassettes as large as 4.5 kb can be exchanged in X-FRMT

(A) J♀ F₁ embryos were injected with FLP cRNA and donor DNA plasmids driving mCherry expression from the 549 bp XOP (Fig. 2) or 3,529 bp XSix6 promoter/enhancer (shown). (B) After stage 45, the surviving embryos were scored for eGFP and/or mCherry expression. The number and percentage of surviving embryos, as well as the total percentage of mCherry-positive embryos is indicated. (C) The genomic DNA from J♀ F₁ tadpoles grown from two uninjected (U1, U2), and two pf1XSix6→mCherryf3 plasmid + FLP cRNA injected (T1, T2) embryos were analyzed by PCR. Primers specifically amplify the host (CarAct-F/eGFP-R1) or donor (Six6-F/mCherry-R2) transgenes (A and C).