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Chapter 3 : Putting Mobile DNA to Work: the Toolbox

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Abstract:

This chapter reviews some mobile DNA tools currently being used in DNA sequencing for manipulating clones efficiently, and in functional analysis of more modest segments of DNA containing one or a few genes as well as entire genomes. It covers the increasing use of transposons as genetic tags and as devices for the delivery and selective destruction of genes. It also provides a brief review of some recent applications of transposons as genetic markers. Two systems have recently been developed to attack the difficulties associated with the often complex subcloning steps required to ensure efficient recombinant protein expression. The original uses of transposons as tools for genome analysis date back to Casadaban’s pioneering work with Mu in . A variation on the footprinting theme for analyzing the genomes of naturally transformable bacteria referred to as genomic analysis and mapping through in vitro transposition (GAMBIT) has also been described. A most powerful technology based on Cre is the activation and shutoff of the target gene in transgenic animals. In recent years, several groups have used mariner/Tc1 elements to perform mutagenesis and to deliver genes to various organisms. The transposon has many attractive features, but its main shortcoming is that it is quite specific for TA dinucleotides in every species examined. This chapter focuses on the practical application of transposons as tangible tools for the manipulation of DNA sequences and of cellular phenotypes.

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3

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Group II Introns
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Figures

Image of Figure 1
Figure 1

In vitro transposition as a method for sequencing DNA. A transposable element (box with black triangles; black triangles represent short terminal inverted repeats recognized by transposase or integrase) bearing a selectable marker and unique priming sites near each end (Primer L and Primer R, arrows) is mixed with the appropriate protein and a target plasmid bearing or DNA segment of interest (bold line). After the transposition reaction occurs in vitro, bacteria expressing markers A and B are selected and DNAs are prepared. DNA from each individual transformant is sequenced with use of primers A and B, resulting in two divergent sequence reads (horizontal arrows) from each transposon which can be linked via the target-site duplication sequence (filled circle) and then assembled with the other sequence reads into a single contig.

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 2

The univector system. A “univector” (pUNI) containing a site (gray triangle) can be combined with one or more host vectors (pHOST) and can be efficiently and precisely recombined using Cre recombinase, generating a family of recombinant plasmids in which YFG is under the control of various promoters (hooked arrows), other 5′ control sequences, and epitope tags (checkerboard). The two plasmids contain compatible origins of replication R6K (gray dot) and Co1E1 (black dot). Adapted from reference .

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 3

The Gateway system. PCR products or restriction fragments containing can be manipulated by a series of steps based on the λ Int system to put its expression under the control of a wide variety of 5′ and/or 3′ regulatory sequences, epitope tags, protein fusions, etc. Boxed triangles represent λ sites, labeled as to type; the triangles are black or gray to indicate type 1 and type 2 sites, respectively. The gene is a gene toxic to conventional strains of generally used for recombinant DNA work. (A) Overall flow of reactions. (B) Steps involved in generating a series of destination or expression clones from a single entry clone. (C) Generation of an entry clone from a PCR product, into which terminal sites have been incorporated. Note that entry clones can also be generated by conventional restriction enzyme/ligase cloning steps. (D) Amino acid sequences that will be added to the N terminus (and, if desired, C terminus) of the Yfg protein. Adapted from reference .

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 4

The Lexicon exon trap retrovirus. An exon trap retrovirus is engineered to contain two reporter genes A and B in orientations opposite to the retrovirus' natural transcription signals. Reporter B is inactive because it lacks a polyadenylation signal. Insertion of the retrovirus into a gene allows reporter B to acquire a polyadenylation signal by splicing (via the splice donor [SD]) signal engineered at the 3′ end of reporter B to adjacent cellular exons, resulting in expression of reporter B. At the same time, expression of is disrupted because of the introduction of the polyadenylation signal upstream of the PGK promoter, which will both incorporate reporter A via the splice donor signal (SA) and truncate the transcript via the polyadenylation signal (pA). Open arrows, LTRs.

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 5

Use of Cre- to activate gene expression. First, a construct consisting of a synthetic “stop” signal (transcriptional terminator) flanked by sites is introduced next to into ES cells by standard homology-dependent gene replacement techniques. Crossing the resultant knockin mouse by a Cre transgenic results in mice in which is activated. Adapted from reference .

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 6

Using Cre- to inactivate gene expression; conditional gene disruption. First, a construct consisting of flanked by sites and, on one side, by a gene and a third site is introduced into ES cells by standard homology-dependent gene replacement techniques. Neo-resistant cell lines are then transiently transfected with a Cre expression construct to eliminate the sequence. This is because the gene and associated enhancer sequences could have undesired effects on the expression of The mice are then bred to homozygosity; because the sites (hopefully) do not interfere with expression, a phenotypically normal mouse is expected. This mouse can then be bred by a strain heterozygous for Δ and containing a transgene driven by a tissue-specific promoter. This leads to excision of the transgene specifically in the target tissue. Adapted from reference . A database of existing Cre-expressing mouse lines may be found at http:// www.mshri.on.ca/nagy/Cre.html.

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Figure 7

Viral vectors for gene therapy. (A) Retroviruses, Ψ, packaging signal; open arrows, LTRs. Retroviral vectors may or may not include a selectable marker gene. Usually the native LTR promoter is used to drive expression, although exogenous promoters can be used ( Fig. 4 ). (B) AAV. ITR, inverted terminal repeat. Exogenous promoters are usually used to drive gene expression in these vectors; minimal promoters are often used to not exceed the packaging limit.

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
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Tables

Generic image for table
Table 1

In vitro transposition system features

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3
Generic image for table
Table 2

Genome-wide mobile DNA-based functional genomics resources, “Security Council” organisms

Citation: Boeke J. 2002. Putting Mobile DNA to Work: the Toolbox, p 24-37. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch3

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