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Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives.

Mátés L, Izsvák Z, Ivics Z - Genome Biol. (2007)

Bottom Line: To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner.Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila.In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str, 13092 Berlin, Germany.

ABSTRACT
To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.

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Related in: MedlinePlus

Summary of the basic gene trapping strategies. Genomic integration of the gene trap markers is facilitated by transposition. (a) Structure of a putative endogenous target gene. (b) The enhancer traps incorporate a reporter expression cassette driven by a minimal promoter (mP) that only results in reporter gene expression when it is affected by a genomic enhancer element, for example by transposition into a gene. (c) The conventional gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a polyadenylation signal (pA). The reporter is only expressed when transcription starts from the promoter of an endogenous transcription unit. Thus, the expression of the reporter follows the expression pattern of the trapped gene. The GAL4 system is a particularly interesting version of gene or enhancer trapping in Drosophila. Here, GAL4 expression is driven by the trapped regulatory regions of endogenous genes in GAL4 driver lines. Using these driver lines, any protein of interest can be over-expressed or mis-expressed by crossing these lines with others carrying the protein of interest expressed from GAL4 controlled promoter (upstream activator sequence [UAS]). (d) Polyadenylation (poly(A)) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a poly(A) signal. Therefore, reporter gene expression depends on splicing to downstream exon(s) of a Pol II transcription unit containing a poly(A) signal. (e) The 'dual tagging' vectors are based on both gene and poly(A) trapping of a targeted transcription unit. (f) The protein trap strategy inserts an artificial exon encoding a reporter into a gene, where the reporter is designed to be incorporated at the protein level into the endogenous gene product. The P element based protein trap (PTT) vector set has been created to tag proteins in all three reading frames with green fluorescent protein (GFP) in Drosophila. (g) Targeted over-expression/mis-expression is a version of the poly(A) trap strategy. Here, a strong promoter (sP) oriented toward the outside of the element is directly followed by a splice donor site. This strategy allows over-expression/mis-expression of truncated or full length endogenous proteins, depending on the site of vector integration. An improved version of this approach is the so-called modular mis-expression system in Drosophila. Here, a GAL4 controlled promoter (UAS) is inserted by the P element into an endogenous transcription unit. This arrangement allows expression of the trapped gene in any arbitrary manner of interest by crossing the carrier line with a GAL4 driver line. E1 to E4, exons 1 to 4; GAGA, GAGA transcription factor (GAF) binding site; ITR, inverted terminal repeat; P, promoter; pA, poly(A).
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Figure 2: Summary of the basic gene trapping strategies. Genomic integration of the gene trap markers is facilitated by transposition. (a) Structure of a putative endogenous target gene. (b) The enhancer traps incorporate a reporter expression cassette driven by a minimal promoter (mP) that only results in reporter gene expression when it is affected by a genomic enhancer element, for example by transposition into a gene. (c) The conventional gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a polyadenylation signal (pA). The reporter is only expressed when transcription starts from the promoter of an endogenous transcription unit. Thus, the expression of the reporter follows the expression pattern of the trapped gene. The GAL4 system is a particularly interesting version of gene or enhancer trapping in Drosophila. Here, GAL4 expression is driven by the trapped regulatory regions of endogenous genes in GAL4 driver lines. Using these driver lines, any protein of interest can be over-expressed or mis-expressed by crossing these lines with others carrying the protein of interest expressed from GAL4 controlled promoter (upstream activator sequence [UAS]). (d) Polyadenylation (poly(A)) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a poly(A) signal. Therefore, reporter gene expression depends on splicing to downstream exon(s) of a Pol II transcription unit containing a poly(A) signal. (e) The 'dual tagging' vectors are based on both gene and poly(A) trapping of a targeted transcription unit. (f) The protein trap strategy inserts an artificial exon encoding a reporter into a gene, where the reporter is designed to be incorporated at the protein level into the endogenous gene product. The P element based protein trap (PTT) vector set has been created to tag proteins in all three reading frames with green fluorescent protein (GFP) in Drosophila. (g) Targeted over-expression/mis-expression is a version of the poly(A) trap strategy. Here, a strong promoter (sP) oriented toward the outside of the element is directly followed by a splice donor site. This strategy allows over-expression/mis-expression of truncated or full length endogenous proteins, depending on the site of vector integration. An improved version of this approach is the so-called modular mis-expression system in Drosophila. Here, a GAL4 controlled promoter (UAS) is inserted by the P element into an endogenous transcription unit. This arrangement allows expression of the trapped gene in any arbitrary manner of interest by crossing the carrier line with a GAL4 driver line. E1 to E4, exons 1 to 4; GAGA, GAGA transcription factor (GAF) binding site; ITR, inverted terminal repeat; P, promoter; pA, poly(A).

Mentions: The P element has been the most widely used vehicle for these purposes. The mutagenicity of P element insertions is higher than that of Tc1/mariner elements (Table 1). Moreover, P elements appear to transpose efficiently with large cargo sequences inserted within the transposon (Table 1). The early mutagenesis screens carried out in Drosophila utilized vectors that harbor marker genes that are easy to screen such as white, and functional bacterial components (antibiotic resistance genes, origins of replication) that aid molecular analysis of the transposon insertion sites. Vectors of later generations were equipped with gene trapping features, representing an improvement to the basic design. The basic strategies employed to enhance the mutagenicity as well as reporting capabilities of insertional vectors by trapping transcription units are shown in Figure 2. Moreover, elements of binary systems for controlled gene expression such as the GAL4 DNA binding transcription factor (GAL4)/GAL4-upstream activator sequence (UAS) system, or for site-directed recombination such as the flip recombinase (FLP)/FLP recombinase target (FRT) system have also been incorporated into advanced vectors. Thus, a range of versatile experimental designs using P elements for insertional mutagenesis has been developed.


Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives.

Mátés L, Izsvák Z, Ivics Z - Genome Biol. (2007)

Summary of the basic gene trapping strategies. Genomic integration of the gene trap markers is facilitated by transposition. (a) Structure of a putative endogenous target gene. (b) The enhancer traps incorporate a reporter expression cassette driven by a minimal promoter (mP) that only results in reporter gene expression when it is affected by a genomic enhancer element, for example by transposition into a gene. (c) The conventional gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a polyadenylation signal (pA). The reporter is only expressed when transcription starts from the promoter of an endogenous transcription unit. Thus, the expression of the reporter follows the expression pattern of the trapped gene. The GAL4 system is a particularly interesting version of gene or enhancer trapping in Drosophila. Here, GAL4 expression is driven by the trapped regulatory regions of endogenous genes in GAL4 driver lines. Using these driver lines, any protein of interest can be over-expressed or mis-expressed by crossing these lines with others carrying the protein of interest expressed from GAL4 controlled promoter (upstream activator sequence [UAS]). (d) Polyadenylation (poly(A)) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a poly(A) signal. Therefore, reporter gene expression depends on splicing to downstream exon(s) of a Pol II transcription unit containing a poly(A) signal. (e) The 'dual tagging' vectors are based on both gene and poly(A) trapping of a targeted transcription unit. (f) The protein trap strategy inserts an artificial exon encoding a reporter into a gene, where the reporter is designed to be incorporated at the protein level into the endogenous gene product. The P element based protein trap (PTT) vector set has been created to tag proteins in all three reading frames with green fluorescent protein (GFP) in Drosophila. (g) Targeted over-expression/mis-expression is a version of the poly(A) trap strategy. Here, a strong promoter (sP) oriented toward the outside of the element is directly followed by a splice donor site. This strategy allows over-expression/mis-expression of truncated or full length endogenous proteins, depending on the site of vector integration. An improved version of this approach is the so-called modular mis-expression system in Drosophila. Here, a GAL4 controlled promoter (UAS) is inserted by the P element into an endogenous transcription unit. This arrangement allows expression of the trapped gene in any arbitrary manner of interest by crossing the carrier line with a GAL4 driver line. E1 to E4, exons 1 to 4; GAGA, GAGA transcription factor (GAF) binding site; ITR, inverted terminal repeat; P, promoter; pA, poly(A).
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Summary of the basic gene trapping strategies. Genomic integration of the gene trap markers is facilitated by transposition. (a) Structure of a putative endogenous target gene. (b) The enhancer traps incorporate a reporter expression cassette driven by a minimal promoter (mP) that only results in reporter gene expression when it is affected by a genomic enhancer element, for example by transposition into a gene. (c) The conventional gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a polyadenylation signal (pA). The reporter is only expressed when transcription starts from the promoter of an endogenous transcription unit. Thus, the expression of the reporter follows the expression pattern of the trapped gene. The GAL4 system is a particularly interesting version of gene or enhancer trapping in Drosophila. Here, GAL4 expression is driven by the trapped regulatory regions of endogenous genes in GAL4 driver lines. Using these driver lines, any protein of interest can be over-expressed or mis-expressed by crossing these lines with others carrying the protein of interest expressed from GAL4 controlled promoter (upstream activator sequence [UAS]). (d) Polyadenylation (poly(A)) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a poly(A) signal. Therefore, reporter gene expression depends on splicing to downstream exon(s) of a Pol II transcription unit containing a poly(A) signal. (e) The 'dual tagging' vectors are based on both gene and poly(A) trapping of a targeted transcription unit. (f) The protein trap strategy inserts an artificial exon encoding a reporter into a gene, where the reporter is designed to be incorporated at the protein level into the endogenous gene product. The P element based protein trap (PTT) vector set has been created to tag proteins in all three reading frames with green fluorescent protein (GFP) in Drosophila. (g) Targeted over-expression/mis-expression is a version of the poly(A) trap strategy. Here, a strong promoter (sP) oriented toward the outside of the element is directly followed by a splice donor site. This strategy allows over-expression/mis-expression of truncated or full length endogenous proteins, depending on the site of vector integration. An improved version of this approach is the so-called modular mis-expression system in Drosophila. Here, a GAL4 controlled promoter (UAS) is inserted by the P element into an endogenous transcription unit. This arrangement allows expression of the trapped gene in any arbitrary manner of interest by crossing the carrier line with a GAL4 driver line. E1 to E4, exons 1 to 4; GAGA, GAGA transcription factor (GAF) binding site; ITR, inverted terminal repeat; P, promoter; pA, poly(A).
Mentions: The P element has been the most widely used vehicle for these purposes. The mutagenicity of P element insertions is higher than that of Tc1/mariner elements (Table 1). Moreover, P elements appear to transpose efficiently with large cargo sequences inserted within the transposon (Table 1). The early mutagenesis screens carried out in Drosophila utilized vectors that harbor marker genes that are easy to screen such as white, and functional bacterial components (antibiotic resistance genes, origins of replication) that aid molecular analysis of the transposon insertion sites. Vectors of later generations were equipped with gene trapping features, representing an improvement to the basic design. The basic strategies employed to enhance the mutagenicity as well as reporting capabilities of insertional vectors by trapping transcription units are shown in Figure 2. Moreover, elements of binary systems for controlled gene expression such as the GAL4 DNA binding transcription factor (GAL4)/GAL4-upstream activator sequence (UAS) system, or for site-directed recombination such as the flip recombinase (FLP)/FLP recombinase target (FRT) system have also been incorporated into advanced vectors. Thus, a range of versatile experimental designs using P elements for insertional mutagenesis has been developed.

Bottom Line: To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner.Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila.In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str, 13092 Berlin, Germany.

ABSTRACT
To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.

Show MeSH
Related in: MedlinePlus