Replication-deficient adenovirus (Ad) vectors are important vehicles for gene expression studies and gene therapy due to their highly efficient expression of transgenes in a variety of cell types. Constructing Ad recombinants can be laborious and time-consuming, requiring several cloning steps to place the transgene into a prokaryotic transfer vector prior to recombination with the Ad vector in Escherichia coli. In order to streamline this process and make it compatible for the high-throughput generation of Ad recombinants, R. Stanton and colleagues at Cardiff University (Cardiff, UK) have devised a novel Ad vector system that avoids conventional cloning by utilizing recombineering to directly insert a transgene into the Ad vector in E. coli without the need for a transfer vector. Recombineering uses λ phage recombinases expressed in the special E. coli strain SW102 to mediate the homologous recombination of DNA stretches as short as 30 basepairs. PCR amplification of the transgene with primers containing sequences homologous to the ends of their AdZ vector allows the direct insertion of the transgene in the desired orientation into the vector when they are cotransfected into the SW102 E. coli cells. Several AdZ vectors are also configured to allow expression of the transgenic proteins with various tags, as well as the conditional expression of potentially toxic genes through the tet repressor system.
(See “Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function” on page 659.)
A Sticky Situation: Molecular CombingMolecular combing is a DNA fiber–stretching technique often combined with immunofluorescent detection for mapping genomic regions, detecting chromosomal rearrangements, or studying DNA replication and genome stability. Typically, a coverslip coated with the octenyl carbon chain product of a gas-phase silanization reaction is dipped into a buffered DNA solution. DNA strand ends bind to the hydrophobic surface in a pH-dependent manner and are stretched as the coverslip is pulled from the solution at a constant speed, producing irreversibly fixed DNA strands aligned in parallel over the surface of the slide. Successful molecular combing depends on the quality and homogeneity of the silanized coverslips, which are not commercially available. Gas-phase silanization of coverslips requires controlled anhydrous conditions and specialized equipment not available to many labs. Researchers in K. Marheineke's lab at the Ecole Normale Supérieure (Paris, France) have developed a simple liquid-phase silanization protocol for coating slides needed for molecular combing. Characterization of slides produced by this method showed better DNA fiber alignment and stretching than traditionally treated slides. The resulting high-quality, ho-mogenously modified surfaces were suitable for detection of replication forks and for FISH, demonstrating that liquid-phase silanization is a simple and accessible alternative for preparing these coverslips.
(See “A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers” on page 649.)
