Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

Abstract

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal beta-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.

Figure 1

Scale drawing of the Fugu rubripes WAGR region encompassing WT1, RCN1 and PAX6 genes depicting the constructs used in this study. (A) Fugu RCN1. (B) PHAFrWT1MYC. (C) Fugu WT1/pFrWTlac.

Figure 2

A Fugu RCN1 transgene is not spliced efficiently in transgenic mice. (A) Diagram of the Fugu RCN1 genomic transgene showing intron sizes. The sizes of the corresponding mouse introns are shown in parentheses. (B–F) RT–PCR analysis of splicing for individual introns of Fugu RCN1 in transgenic mice. (B) Intron 1, (C) intron 2, (D) intron 3, (E) intron 4, (F) intron 5. Total RNA prepared from kidney of transgenic Fugu RCN1 mice was subjected to RT–PCR across individual intron–exon boundaries. Lane b, H₂O blank. (G and H) RT–PCR analysis of the endogenous Fugu RCN1 RNA in pufferfish kidney. Lane d, DNA amplification; negative control (–) consisting of an equivalent amount of the total RNA preparation subjected to PCR without reverse transcription; PCR of reverse-transcribed RNA (+). RT–PCR products corresponding to spliced and unspliced introns are labelled s and u, respectively.

Figure 3

Efficient splicing of a Fugu rubripes transgene in zebrafish but not in mice. (A) Diagram of pHAFrWT1MYC showing intron sizes, with the sizes of the corresponding mouse introns in parentheses. The asterisk refers to the location of the three amino acid alternative splicing event. Note that fish do not possess an equivalent to mammalian WT1 exon 5. Consequently, Fugu WT1 exon 6 corresponds to mammalian exon 7 and so on. (B) RT–PCR analysis of transgenic zebrafish pools, ZF14 and ZF28, and individual transgenic mouse embryos, M10 and M14, selected to represent low and high expressors respectively. RT–PCR was performed on total RNA isolated from whole embryos using oligonucleotides situated in exon 7 and the myc tag. The negative control consists of an equivalent amount of the total RNA preparation subjected to PCR without reverse transcription, b = H₂O blank. RT–PCR products corresponding to each partly spliced transcript are labelled. (C) RT–PCR analysis of ZF28 and M10 RNA from exon 7 to MYC showing the emergence of each class of PCR product from 24 to 30 cycles. An equivalent amount of RNA was amplified for 30 cycles without reverse transcription as a control (–RT), b = H₂O blank. (D) Analysis of alternative splicing of the transgene in pools of transgenic zebrafish (ZF14 and ZF28) harbouring pHAFrWT1MYC. RT–PCR was performed with oligonucleotides situated in exon 8 and the myc tag. The negative control (–) consists of an equivalent amount of the total RNA preparation subjected to PCR without reverse transcription.

Figure 4

Expression of full-length protein from a pufferfish transgene in zebrafish. (A) Diagram of the genomic Fugu WT1 fragment with C-terminal β-galactosidase fusion. (B) Expression of endogenous WT1 in control 12 h.p.f. zebrafish by in situ hybridisation. (C) X-gal-stained transgenic zebrafish at ∼12 h.p.f. Correct splicing of the construct is evident from X-gal staining resulting from production of a fusion protein. The mosaicism associated with zebrafish transgenesis results in staining only in one half of the pronephric field. (D and E) Examples of mosaicism observed in pronephric expression of Fugu WT1–lacZ fusion protein between individual fish. Arrowheads point to transgene expression (X-gal staining) in the developing pronephos.