Rearranging the centromere of the human Y chromosome with φC31 integrase

We have investigated the ability of the integrase from the Streptomyces φC31 ‘phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a φC31 integrase attB site and the chicken genome

RMDAP: A Versatile, Ready-To-Use Toolbox for Multigene Genetic Transformation

Background: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed. Methodology/Principal Findings: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-ofprinciple experiment was confirmed by successfully transferring several heterologous genes into the plant genome. Conclusions/Significance: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call ‘‘one-sto

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

A mini-chromosome with a functional site is stable in DT40 cells in the presence of φC31 integrase

<p><b>Copyright information:</b></p><p>Taken from "Rearranging the centromere of the human Y chromosome with φC31 integrase"</p><p>Nucleic Acids Research 2005;33(19):6101-6113.</p><p>Published online 24 Oct 2005</p><p>PMCID:PMC1266074.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Stability of mini-chromosomes in clone CA4 inter-chromatid mini-chromosome clone 4 analysed using FISH with probes for the alphoid DNA and the DYZ5 sequence after the indicated time in culture in the presence and absence of selection with puromycin. Zeocin, selecting for integrase expression, was present in both of the cultures. The filled bars indicate the proportion of cells containing a mini-chromosome and the empty bars indicate the proportion of cells lacking a mini-chromosome. () Mini-chromosomes in clone 4 remained structurally intact after four weeks in culture as judged by pulsed field electrophoresis. () Diagrammatic representation of the site-specific recombination reaction between the site in the mini-chromosomes in clone number 4 and a plasmid containing and a promoterless gene conferring resistance to the antibiotic G418; φCneo. () sites on mini-chromosomes in clone number 4 are able to undergo site-specific recombination after one month in culture as judged by PCR. Cells from clone 4 containing the inter-chromatid mini-chromosome were electroporated with the plasmid φCneo and G418 resistant clones isolated. DNA extracted from 12 such clones was analysed by PCR across the products of the site-specific recombination reaction between φCneo and the resident site

Establishing the identity of the φC31 integrase mediated chromosome rearrangements by FISH and by restriction site mapping

<p><b>Copyright information:</b></p><p>Taken from "Rearranging the centromere of the human Y chromosome with φC31 integrase"</p><p>Nucleic Acids Research 2005;33(19):6101-6113.</p><p>Published online 24 Oct 2005</p><p>PMCID:PMC1266074.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () FISH analysis of the inter-chromatid maxi-chromosome in the cell line CA4 clone 6 using probes against the alphoid DNA (red) and the telomere of the short arm of the human Y chromosome () (cY29) (green). () FISH analysis of the inter-chromatid mini-chromosome in the puromycin resistant cell line mini-4 containing an inter-chromatid mini-chromosome using probes against the alphoid DNA (red) and the DYZ5 sequence (green). () FISH analysis of an un-rearranged chromosome (in the cell line XP4) using probes against the alphoid DNA (red) and the DYZ5 sequence (green). The chromosomes in the CA4 cell line are similar. The image of the mini-chromosome is slightly confusing because the two alphoid signals (red), deriving from the sister chromatids, appear to be more centrally located than the DYZ5 sequence and to be a more obvious doublet. The well resolved doublet is a characteristic of the telomeric position of the sequences but the fact that they appear in a sub-telomeric position may reflect the way the chromosome is folded. This distribution of the two sequences was observed consistently. () FISH analysis of the circular deletion mini-chromosome in the cell line XP4 DEL 14 using probes against the alphoid DNA (red) and the DYZ5 sequence (green) of chromosomes. () Arrangement of PmeI sites around the alphoid and DYZ5 sequences in the cell lines, CA4 and CA4 clone 15 following φC31 integrase mediated intra-chromatid inversion of the interval between the and sites. () Filter hybridization and restriction enzyme analysis of the arrangement of the PmeI sites around the alphoid and DYZ5 sequences in the cell lines ΔΔ16, CA4 and CA4 clone 15 that was isolated after introduction of a φC31 integrase expression construct in the absence of puromycin

Deleting the centromere of the human mini-chromosome XP4 with the φC31 integrase

<p><b>Copyright information:</b></p><p>Taken from "Rearranging the centromere of the human Y chromosome with φC31 integrase"</p><p>Nucleic Acids Research 2005;33(19):6101-6113.</p><p>Published online 24 Oct 2005</p><p>PMCID:PMC1266074.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Assay of site-specific recombination around the puromycin resistance and genes by restriction enzyme digestion, gel electrophoresis and filter hybridization following stable expression of 3′-NLS φC31 integrase. () Assay by PCR of , , and sites in the clones analysed in A. () Stability of mini-chromosomes in clone XP4 derived deletion clone DEL 14 analysed using FISH with probes for the alphoid DNA and the DYZ5 sequence after the indicated time in culture. The circular mini-chromosome could be readily discriminated from the un-rearranged chromosome on the basis of size and more easily by the intensity of staining with the DYZ5 probe (as indicated in )