Chapter 3 : Putting Mobile DNA to Work: the Toolbox

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 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.

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.

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.