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Generating and manipulating transgenic animals using transposable elements.

Largaespada DA - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Transposable elements, or transposons, have played a significant role in the history of biological research.The sophistication of these applications and the number of active elements are likely to increase over the next several years.General considerations and predictions about the future utility of transposon technology are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA. larga002@tc.umn.edu

ABSTRACT
Transposable elements, or transposons, have played a significant role in the history of biological research. They have had a major influence on the structure of genomes during evolution, they can cause mutations, and their study led to the concept of so-called "selfish DNA". In addition, transposons have been manipulated as useful gene transfer vectors. While primarily restricted to use in invertebrates, prokaryotes, and plants, it is now clear that transposon technology and biology are just as relevant to the study of vertebrate species. Multiple transposons now have been shown to be active in vertebrates and they can be used for germline transgenesis, somatic cell transgenesis/gene therapy, and random germline insertional mutagenesis. The sophistication of these applications and the number of active elements are likely to increase over the next several years. This review covers the vertebrate-active retrotransposons and transposons that have been well studied and adapted for use as gene transfer agents. General considerations and predictions about the future utility of transposon technology are discussed.

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"Copy-and-paste" and "cut-and-paste" transposons have been adapted for use as gene transfer vectors. In the top half of the figure, transposition of naturally occurring transposons is depicted. In the lower half of the figure, the general methods used to adapt these transposons for use as gene transfer agents is shown. Direct terminal repeats (TR) flank some retrotransposons. Inverted terminal repeats (IR) flank cut and paste transposons. Retrotransposons, such as the L1 element, encode open reading frames (ORF) of unknown function as well as integrases (IN) and reverse transcriptases (RT). Both kinds of elements can be manipulated so that special vector sequences are inserted. In the case of retrotransposons, the vector sequences are inserted into the 3' untranslated region. In the case of the "cut and paste", DNA transposons, the vector sequences replace the transposase gene, which is expressed from a heterologous promoter in trans.
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Figure 1: "Copy-and-paste" and "cut-and-paste" transposons have been adapted for use as gene transfer vectors. In the top half of the figure, transposition of naturally occurring transposons is depicted. In the lower half of the figure, the general methods used to adapt these transposons for use as gene transfer agents is shown. Direct terminal repeats (TR) flank some retrotransposons. Inverted terminal repeats (IR) flank cut and paste transposons. Retrotransposons, such as the L1 element, encode open reading frames (ORF) of unknown function as well as integrases (IN) and reverse transcriptases (RT). Both kinds of elements can be manipulated so that special vector sequences are inserted. In the case of retrotransposons, the vector sequences are inserted into the 3' untranslated region. In the case of the "cut and paste", DNA transposons, the vector sequences replace the transposase gene, which is expressed from a heterologous promoter in trans.

Mentions: Many excellent reviews have been written on the subject of transposable elements and it is not the goal of this review to systematically cover that very large field, which really would require a textbook to do the subject justice [1-3]. It can be said, however, that transposons come in two general types (Figure 1). The "copy and paste" retrotransposons are mobilized by transcribing an RNA copy, that then becomes reverse transcribed and is integrated elsewhere in the genome. In contrast, the "cut and paste" transposable elements transpose by the direct excision from DNA and insertion elsewhere in the genome. Both types of elements have now been used in vertebrate cell lines and animals as gene transfer vectors. Retrotransposons come in many classes and include retroviruses, which will not be considered in this review. Retrotransposon elements require at a minimum, an internal promoter and coding sequences for an integrase and reverse transcriptase protein. Many of these elements encode proteins that act primarily in cis, on the RNA transcript from which they were translated. Thus, in biotechnology applications, foreign gene sequences must be added to the 3' untranslated region (UTR) of the retrotransposon vector (Figure 1). In contrast, the cut and paste transposases can act in trans. These elements have an internal promoter and encode a single protein "transposase" which binds to terminal repeat sequences on the ends of the element, causing it to be excised and inserted elsewhere. Because the cut and paste transposons can act in trans, it is feasible to supply the transposase by a variety of methods (DNA, RNA and possibly transferred protein) and insert any desired sequence in the transposon vector DNA itself (Figure 1). These sequences might include genes for germline or somatic cell transgenesis, sequences for insertional mutagenesis, or recognition by site-specific recombinases.


Generating and manipulating transgenic animals using transposable elements.

Largaespada DA - Reprod. Biol. Endocrinol. (2003)

"Copy-and-paste" and "cut-and-paste" transposons have been adapted for use as gene transfer vectors. In the top half of the figure, transposition of naturally occurring transposons is depicted. In the lower half of the figure, the general methods used to adapt these transposons for use as gene transfer agents is shown. Direct terminal repeats (TR) flank some retrotransposons. Inverted terminal repeats (IR) flank cut and paste transposons. Retrotransposons, such as the L1 element, encode open reading frames (ORF) of unknown function as well as integrases (IN) and reverse transcriptases (RT). Both kinds of elements can be manipulated so that special vector sequences are inserted. In the case of retrotransposons, the vector sequences are inserted into the 3' untranslated region. In the case of the "cut and paste", DNA transposons, the vector sequences replace the transposase gene, which is expressed from a heterologous promoter in trans.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC280724&req=5

Figure 1: "Copy-and-paste" and "cut-and-paste" transposons have been adapted for use as gene transfer vectors. In the top half of the figure, transposition of naturally occurring transposons is depicted. In the lower half of the figure, the general methods used to adapt these transposons for use as gene transfer agents is shown. Direct terminal repeats (TR) flank some retrotransposons. Inverted terminal repeats (IR) flank cut and paste transposons. Retrotransposons, such as the L1 element, encode open reading frames (ORF) of unknown function as well as integrases (IN) and reverse transcriptases (RT). Both kinds of elements can be manipulated so that special vector sequences are inserted. In the case of retrotransposons, the vector sequences are inserted into the 3' untranslated region. In the case of the "cut and paste", DNA transposons, the vector sequences replace the transposase gene, which is expressed from a heterologous promoter in trans.
Mentions: Many excellent reviews have been written on the subject of transposable elements and it is not the goal of this review to systematically cover that very large field, which really would require a textbook to do the subject justice [1-3]. It can be said, however, that transposons come in two general types (Figure 1). The "copy and paste" retrotransposons are mobilized by transcribing an RNA copy, that then becomes reverse transcribed and is integrated elsewhere in the genome. In contrast, the "cut and paste" transposable elements transpose by the direct excision from DNA and insertion elsewhere in the genome. Both types of elements have now been used in vertebrate cell lines and animals as gene transfer vectors. Retrotransposons come in many classes and include retroviruses, which will not be considered in this review. Retrotransposon elements require at a minimum, an internal promoter and coding sequences for an integrase and reverse transcriptase protein. Many of these elements encode proteins that act primarily in cis, on the RNA transcript from which they were translated. Thus, in biotechnology applications, foreign gene sequences must be added to the 3' untranslated region (UTR) of the retrotransposon vector (Figure 1). In contrast, the cut and paste transposases can act in trans. These elements have an internal promoter and encode a single protein "transposase" which binds to terminal repeat sequences on the ends of the element, causing it to be excised and inserted elsewhere. Because the cut and paste transposons can act in trans, it is feasible to supply the transposase by a variety of methods (DNA, RNA and possibly transferred protein) and insert any desired sequence in the transposon vector DNA itself (Figure 1). These sequences might include genes for germline or somatic cell transgenesis, sequences for insertional mutagenesis, or recognition by site-specific recombinases.

Bottom Line: Transposable elements, or transposons, have played a significant role in the history of biological research.The sophistication of these applications and the number of active elements are likely to increase over the next several years.General considerations and predictions about the future utility of transposon technology are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA. larga002@tc.umn.edu

ABSTRACT
Transposable elements, or transposons, have played a significant role in the history of biological research. They have had a major influence on the structure of genomes during evolution, they can cause mutations, and their study led to the concept of so-called "selfish DNA". In addition, transposons have been manipulated as useful gene transfer vectors. While primarily restricted to use in invertebrates, prokaryotes, and plants, it is now clear that transposon technology and biology are just as relevant to the study of vertebrate species. Multiple transposons now have been shown to be active in vertebrates and they can be used for germline transgenesis, somatic cell transgenesis/gene therapy, and random germline insertional mutagenesis. The sophistication of these applications and the number of active elements are likely to increase over the next several years. This review covers the vertebrate-active retrotransposons and transposons that have been well studied and adapted for use as gene transfer agents. General considerations and predictions about the future utility of transposon technology are discussed.

Show MeSH
Related in: MedlinePlus