October 21, 1999;
A gene trap approach in Xenopus.
The frog transgenesis technique ultimately promises to make mutagenesis possible through random insertion of plasmid DNA into the genome. This study was undertaken to evaluate whether a gene trap approach combined with transgenesis would be appropriate for performing insertional mutagenesis in Xenopus embryos. Firstly, we confirmed that the transgenic technique results in stable integration into the genome and that transmission through the germline occurs in the expected Mendelian fashion. Secondly, we developed several gene trap vectors, using the green fluorescent protein (GFP) as a marker. Using these vectors, we trapped several genes in Xenopus laevis that are expressed in a spatially restricted manner, including expression in the epiphysis
, the olfactory bulb
and placodes, the eyes
, muscles, tail
. Finally, we cloned one of the trapped genes using 5'' rapid amplification of cDNA ends polymerase chain reaction (RACE
PCR). These results suggest that the transgenic technique combined with a gene trap approach might provide a powerful method for generating mutations in endogenous genes in Xenopus.
[+] show captions
Figure 1. Germline transmission of the γ1crystallin�GFP transgene in X. laevis. (a) Image of four F1 stage 30 embryos from transgenic line 1. Note that two out of the four embryos express GFP in the lens. (b) Metamorphosed F1 froglet expressing GFP in the lens.
Figure 2. Gene trap embryos expressing GFP in different tissues. The top left panel shows a side view and ventral view of two tadpoles. The colored boxes outline the regions and orientation used to take each picture. (a,b) SA1 and SA2 were generated using the SAGT vector. GFP expression in SA1 was ubiquitous. (a) A magnified view of the head from SA1. Note strong expression in the lens. (b) View of the tail from SA2. Note strong expression in the somites. (c-e) SEδ1, 2 and 3 were three separate embryos containing the SEδGT gene trap vector. Each embryo expressed GFP in a distinct manner. (c) SEδ1 expressed GFP in the inner ear (otoconia; arrow), (d) SEδ2 expressed GFP in neurons flanking the spinal cord and (e) SEδ3 expressed GFP in the coiled intestine. The white dots outline the eye in (c).
Figure 3. Gene trap embryos expressing the GFP within the brain. (a) Dorsal view of the head of a transgenic embryo containing a GFP fusion under the control of the neural β-tubulin promoter (NBT-GFP). GFP expression is observed throughout the brain, including the cranial nerves. This embryo serves as a reference for positioning the expression of the different gene trap embryos along the anterior-posterior axis. The colored boxes outline the regions of the head photographed in (b, yellow), (c, blue) and (d, red). (b) ET1 was generated using the exon trap vector ET. GFP fluorescence was restricted to the olfactory placodes, nerves and bulbs. (c) SE1 was generated using the SEGT gene trap vector and expressed GFP in the pineal gland (arrow) and the midbrain-hindbrain junction (asterisk in panels (a), (c) and (d)). (d) SA2 was generated using the SAGT gene trap vector. Expression was observed weakly throughout the brain.
Figure 4. Cloned gene trap sequences. (a) Nucleotide and predicted amino acid sequences of the Engrailed 2/gly-GFP junction in the SEGT gene trap vector. A vertical arrow indicates the splice site. The Engrailed 2 SA sequence is shown in blue with the intron in lowercase letters and the exon in uppercase letters. The glycine bridge is shown in purple and GFP in green. (b) Sequence obtained from a trapped gene obtained using a 5′ RACE PCR approach (shown in pink). The predicted amino acid sequence is indicated underneath the nucleotide sequence.