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Category: Microbial Genetics and Molecular Biology
Putting Mobile DNA to Work: the Toolbox, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap03-1.gif /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap03-2.gifAbstract:
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 Escherichia coli. 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.
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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 YFGor 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.
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 YFGor 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.
The univector system. A “univector” (pUNI) containing a loxPsite (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 44 .
The univector system. A “univector” (pUNI) containing a loxPsite (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 44 .
The Gateway system. PCR products or restriction fragments containing YFGcan 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 attsites, labeled as to type; the triangles are black or gray to indicate type 1 and type 2 sites, respectively. The ccdBgene is a gene toxic to conventional strains of E. coligenerally 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 YFG PCR product, into which terminal attB 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 33 .
The Gateway system. PCR products or restriction fragments containing YFGcan 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 attsites, labeled as to type; the triangles are black or gray to indicate type 1 and type 2 sites, respectively. The ccdBgene is a gene toxic to conventional strains of E. coligenerally 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 YFG PCR product, into which terminal attB 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 33 .
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 YFG 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 YFG transcript via the polyadenylation signal (pA). Open arrows, LTRs.
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 YFG 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 YFG transcript via the polyadenylation signal (pA). Open arrows, LTRs.
Use of Cre-lox to activate gene expression. First, a construct consisting of a synthetic “stop” signal (transcriptional terminator) flanked by lox sites is introduced next to YFG into ES cells by standard homology-dependent gene replacement techniques. Crossing the resultant knockin mouse by a Cre transgenic results in mice in which YFG is activated. Adapted from reference 60 .
Use of Cre-lox to activate gene expression. First, a construct consisting of a synthetic “stop” signal (transcriptional terminator) flanked by lox sites is introduced next to YFG into ES cells by standard homology-dependent gene replacement techniques. Crossing the resultant knockin mouse by a Cre transgenic results in mice in which YFG is activated. Adapted from reference 60 .
Using Cre-lox to inactivate gene expression; conditional gene disruption. First, a construct consisting of YFG flanked by lox sites and, on one side, by a neo gene and a third lox 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 neo sequence. This is because the neo gene and associated enhancer sequences could have undesired effects on the expression of YFG. The mice are then bred to homozygosity; because the lox sites (hopefully) do not interfere with expression, a phenotypically normal mouse is expected. This mouse can then be bred by a strain heterozygous for ΔYFG and containing a cre transgene driven by a tissue-specific promoter. This leads to excision of the transgene specifically in the target tissue. Adapted from reference 60 . A database of existing Cre-expressing mouse lines may be found at http:// www.mshri.on.ca/nagy/Cre.html.
Using Cre-lox to inactivate gene expression; conditional gene disruption. First, a construct consisting of YFG flanked by lox sites and, on one side, by a neo gene and a third lox 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 neo sequence. This is because the neo gene and associated enhancer sequences could have undesired effects on the expression of YFG. The mice are then bred to homozygosity; because the lox sites (hopefully) do not interfere with expression, a phenotypically normal mouse is expected. This mouse can then be bred by a strain heterozygous for ΔYFG and containing a cre transgene driven by a tissue-specific promoter. This leads to excision of the transgene specifically in the target tissue. Adapted from reference 60 . A database of existing Cre-expressing mouse lines may be found at http:// www.mshri.on.ca/nagy/Cre.html.
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.
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.
In vitro transposition system features
In vitro transposition system features
Genome-wide mobile DNA-based functional genomics resources, “Security Council” organisms
Genome-wide mobile DNA-based functional genomics resources, “Security Council” organisms