Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the λ integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe–plant interactions.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.

Handoff from recombinase to replisome: Insights from transposition

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRFα2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB–DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRFα2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRFα2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.

Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice.

Site-specific recombinases are being developed as tools for "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. Here it is demonstrated that Flp recombinase can effectively mediate site-specific excisional recombination in mouse embryonic stem cells, in differentiating embryonal carcinoma cells, and in transgenic mice. Broad Flp expression is compatible with normal development, suggesting that Flp can be used to catalyze recombination in most cell types. These properties indicate that Flp can be exploited to make prescribed alterations in the mouse genome.

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.