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. 2012 Sep 18;109(38):15389-94.
doi: 10.1073/pnas.1206131109. Epub 2012 Aug 20.

Conditional control of gene function by an invertible gene trap in zebrafish

Terri T Ni  1 , Jianjun Lu, Meiying Zhu, Lisette A Maddison, Kelli L Boyd, Lindsey Huskey, Bensheng Ju, Daniel Hesselson, Tao P Zhong, Patrick S Page-McCaw, Didier Y Stainier, Wenbiao Chen
Affiliations

Conditional control of gene function by an invertible gene trap in zebrafish

Terri T Ni et al. Proc Natl Acad Sci U S A. .

Abstract

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The FT1 mutagen functions regardless of the starting orientation and the trap is invertible with both the cre and flp recombinases. The FT1 mutagen consists of a FlEx-based invertible cassette that consists of lox sites (brackets) and frt sites (braces) that allow stable inversion of the cassette with cre and flp, respectively. The invertible cassette carries an mCherry reporter exon (red rectangle) downstream of a strong splice acceptor and upstream of five repeats of a transcriptional stop and polyadenylation sequence derived from the BGH gene (red octagon). This cassette is carried on a Tol2 transposon to facilitate transgenesis (double-headed gray arrow). (A) Insertion of the FT1 mutagen into an intron in the gene-trapping orientation (T) results in splicing from the endogenous 5′ exon to the strong splice acceptor and generation of an mCherry fusion transcript that possibly produces a fusion protein, transcriptional termination, and potentially, loss-of-function of the endogenous gene. The FlEx cassette may be inverted with either tissue-specific expression of cre or flp resulting in tissue-specific rescue (Left) or flp/cre mRNA injection at the one-cell stage, resulting in global inversion and rescue (Right). The cassette may be inverted one additional time (Bottom). (B) Insertion of the FT1 mutagen in the neutral (nontrapping) orientation (N). The gene trap cassette is not active in this orientation, allowing transcriptional read through and causing little effect on expression of the endogenous gene. The FlEx cassette may be inverted with either tissue-specific expression of cre/flp, resulting in tissue-specific gene knockout (KO) (Left) or flp/cre mRNA injection resulting in global KO. An additional cassette inversion can be performed for tissue-specific or global inversion of the cassette and subsequent rescue. Although cre and flp function equivalently for inversion in germline and somatic cells, only cre-mediated inversion in somatic cell is depicted for simplicity.
Fig. 2.
Fig. 2.
Mutagenicity and interconversion of T and N alleles. (A) Bar graph (mean ± SD) of qRT-PCR data showing the transcript levels of the affected genes in homozygotes relative to wild-type siblings at 3.5 dpf. The mutagenic state of the insertions is indicated at the Top of the graph. (B) Bar graph (mean ± SD) of qRT-PCR data showing the transcript levels of affected genes in 3.5-dpf homozygotes of the insertion alleles with Tg(hsp70l:Cre)VU297 relative to homozygotes without Tg(hsp70l:Cre)VU297. The mutagenic state of the insertions is indicated at the Top of the graph. Embryos were heat shocked at 38.5 °C for 40 min starting at 9 hpf.
Fig. 3.
Fig. 3.
Phenotypes of supv3l1GtT/GtT mutants. (A) Gross morphological defects at 5 dpf in supv3l1Gt/Gt showing smaller eyes, lack of an inflated swim bladder, small and dark liver (arrowhead), and underdeveloped and dark intestine (arrow). (B) RT-PCR analysis showing undetectable supv3l1 mRNA in the mutant (Upper ) with normal β-actin mRNA levels (Lower). (C) Bar graph (mean ± SE) of whole-animal Western blot analysis of the levels of components of mitochondrial complexes showing a decrease in the levels of NDUFB6 (complex I), CoxVa (complex IV), and F-ATPase (complex V) in the mutants (n = 4). P < 0.05 for complex IV and ATPase. The difference for complex I did not reach statistical significance (P = 0.12). (D) Bar graph (mean ± SEM) showing a decrease in mitochondrial DNA relative to nuclear DNA (β-actin 2) (n = 4). P < 0.05 for both ND2 and ND6. n = 4.
Fig. 4.
Fig. 4.
Liver-specific expression of supv3l1 in supv3l1GtT/GtT mutants rescues hepatocyte defects. (A–C) Images of Lipan (A), LiPan/supv3l1GtT/GtT (B), LiPan/hepatocyte-Cre+/−/supv3l1GtT/GtT (C) showing liver agenesis in supv3l1GtT/GtT and liver-specific rescue by hepatocyte-Cre. Other defects in supv3l1GtT/GtT remain in hepatocyte-Cre+/−/supv3l1GtT/GtT. Each image is a merge of a bright-field image and a red fluorescent image of the same larva. (D–F) In situ hybridization analysis of transferrin a mRNA expression in 5-dpf larvae showing the specific expression in the liver of wild-type and heterozygous larva (90/114) (D), the absence in supv3l1GtT/GtT (12/114) (E), and the restoration in the liver-specific rescue hepatocyte-Cre+/−/supv3l1GtT/GtT (12/114) (F). Eye size in E and F remains smaller than in D. Observed numbers are not different from expectation (χ2 test, P = 0.623). (G and H) Liver H&E staining of wild-type (G), supv3l1GtT/GtT (H), and hepatocyte-Cre+/−/supv3l1GtT/GtT (I) larvae at 5 dpf, indicating the poorly differentiated hepatocytes with vacuolated cytoplasm and weak nuclear staining in global mutants (H) is fully restored by hepatocyte-Cre (I). Arrowheads indicate hepatocytes.
Fig. 5.
Fig. 5.
Phenotype of liver-specific supv3l1 inactivation. (A–D) Confocal images of the Left (A and B) and Right (C and D) sides of 21-d-old LiPan (A and C) and LiPan/supv3l1LKO (B and D) fish showing detached hepatocytes (white arrows) (B and D) and smaller right liver lobe (white arrow) in supv3l1LKO fish (D). (E and F) Histological analysis of wild-type (E) and supv3l1LKO (F) fish indicate disorganized liver with dead cells (arrowhead) but normal intestinal lumen (black arrows) in supv3l1LKO fish (F). (F) Survival curve of supv3l1LKO fish (lens YFP+) compared with supv3l1GtNf/GtNf siblings (lens YFP−) during the first 6 wk of life. One group was fed a larval diet until 28 dpf followed by a juvenile diet (red) and the other group was kept on the larval diet (blue). (G) Photograph of a wild-type and a supv3l1LKO mutant at 5 wk of age showing the smaller size of the mutant.
See this image and copyright information in PMC

Comment in

  • The changing conditions of zebrafish mutants.
    Burgess SM. Burgess SM. Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15082-3. doi: 10.1073/pnas.1212832109. Epub 2012 Sep 4. Proc Natl Acad Sci U S A. 2012. PMID: 22949684 Free PMC article. No abstract available.

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