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Review
, 33 (11), 784-801

Transposons As Tools for Functional Genomics in Vertebrate Models

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Review

Transposons As Tools for Functional Genomics in Vertebrate Models

Koichi Kawakami et al. Trends Genet.

Abstract

Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats.

Keywords: animal models; forward genetics; genetic screens; genomics; insertional mutagenesis; stem cells.

Figures

Figure 1
Figure 1. Mechanism of cut-and-paste transposition
The transposon is a self-contained mobile genetic element containing a transposase coding sequence (orange box) flanked by terminal inverted repeats (TIRs; thick black arrows on the left and right). Transcriptional regulatory elements, including a promoter (small arrow) and polyA site (light blue box) regulate the expression of the transposase (orange spheres). Transposase molecules bind to the TIRs and catalyze the movement of the transposase to a new chromosomal location.
Figure 2
Figure 2. Possible mutagenic consequences of transposon integration in or close to a transcription unit
(A) The figure depicts a hypothetical transcription unit with a promoter (red arrow), three exons (yellow boxes) and a polyA signal (pA, light blue box). Splicing is indicated by dashed lines between the exons. (B) Transposition into an exon disrupts the coding sequence of the gene. Transposition into an intron may result in expression of a sense (C) or antisense (D) transcript from the transposon’s promoter. (E) Cryptic splice sites present in the transposon sequence may result in the generation of truncated transcripts. (F) Transposon insertions at the 5′-transcriptional regulatory region of a gene may introduce an alternative transcription start site or may override the transcriptional program of the gene’s endogenous promoter. (G) Transposon insertion in the 3′-UTR of a gene may introduce an alternative pA.
Figure 3
Figure 3. Mutagenic cassettes
(A) A hypothetical transcription unit is depicted with an upstream promoter (red arrow), three exons (yellow boxes) and a polyadenylation signal (pA). An intronic transposon insertion is typically not mutagenic, because the transposon is spliced out from the primary RNA transcript together with the targeted intron sequences, thereby resulting in normal gene expression. (B) Gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a pA. The SA truncates the transcript, and expression of the reporter follows the expression pattern of the trapped gene. (C) Poly(A) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a pA signal. Therefore, reporter gene expression depends on splicing to downstream exon/s of a Pol II transcription unit containing a pA. (D) The oncogene trap contains SA signals in both orientations and a bidirectional pA signal to disrupt transcription, as well as a strong, viral enhancer/promoter (thick orange arrow) that drives transcription towards the outside of an inserted transposon, and thereby overexpresses a gene product. In case this transposon lands in the 5′ transcriptional regulatory region of a gene (E), a full-length transcript might be overexpressed.
Figure 4
Figure 4. Experimental mobilzation of transposons
(A) Plasmid-to-genome mobilization. The transposon is typically mobilized out of transfected plasmids upon transient expression of the transposase. (B) Intra-genomic mobilization. Upon transposase expression, the genomically located transposon will be excised from the donor site and re-integrate at a different genomic location.
Figure 5
Figure 5. Generation of knock-out animals by insertional mutagenesis in embryonic and spermatogonial stem cells
Cultured embryonic stem cells (ESCs) or spermatogonial stem cell (SSCs) are transfected with mutagenic transposon and transposase constructs that will lead to thousands of transposon insertions covering all chromosomes. Those cells, in which insertions occurred in genes can be selected based on activation of a reporter, and the insertion sites can be mapped. Clonally derived ESCs are transplanted into mouse embryos that will develop into chimeric animals that need to be crossed with wild-type (WT) animals to derive F1 heterozygotes. An F1 intercross will yield F2 homozygotes, in which a phenotype can be studied. SSC clones or polyclonal insertion libraries can be directly transplanted into the testes of sterile males, in which the spermatogonial step cells will undergo spermatogenesis. These transplanted males are crossed with WT females to pass the insertions through the germline and generate transgenic/knock-out animals.
Figure 6
Figure 6. A scheme for gene and enhancer trapping for the Gal4-UAS system
A gene trap or an enhancer trap construct containing gal4 is injected into fertilized eggs with the transposase mRNA. Injected founder fish are raised and mated with UAS:GFP reporter fish. When gal4 is expressed under the control of an endogenous promoter/enhancer, GFP expression is induced in the F1 embryos via the Gal4-UAS system. Double transgenic F1 embryos express GFP in spatially and temporally restricted fashions may be picked up.
Figure 7
Figure 7. Transgenic fish with specific gal4/gfp expression patterns
(A) Blood vessels in gSAIzGFF478A embryos at 3 days post-fertilization (dpf) [108]. (B) Central nervous system (cerebellum) in SAGFF128A embryo at 5 dpf [109]. (C) Central and enteric nervous system in SAGFF234A at 5 dpf [110]. (D) Lateral line glial cells in gSAGFF202A at 5 dpf [111]. (E) Caudal trunk (and the wound epidermis) in HGn21A embryo at 1 dpf [112]. (F) Fgf7b-positive muscle cells in gSAIzGFFD164A at 1 dpf [113]. (G) Central nervous system and motor neurons in the spinal cord in SAIGFF213A at 1 dpf [114]. (H) Calcium imaging of the pretectal neurons while a larva recognizes a paramecia (prey) [115].

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