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Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector.

Erratum to: “I-SceI meganuclease mediates highly efficient transgenesis in fish” Mechanisms of Development, Volume 120, Issue 2, February 2003, Pages 267; The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

International audience; The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

prp6 and prp9 thermosensitive (ts) mutants are affected in pre-mRNA splicing and transport from the nucleus to the cytoplasm. PRP6 and PRP9 wild-type alleles have been sequenced. DNA sequence analysis reveals homologies in the 5' and 3' non-coding regions, suggesting a common regulation of gene expression. PRP6 and PRP9 genes encode a 899 amino acid and a 530 amino acid protein, respectively. The PRP6 protein has repeated motifs that evoke helix-loop-helix structures. Both PRP6 and PRP9 proteins have cysteine/histidine motifs loosely related to those found in zinc finger proteins. The substitution of some, but not all, of these residues by directed mutagenesis has a critical effect on the protein function. Homology searches reveal that two other proteins known to be involved in the nuclear splicing pathway--the yeast PRP11 and the human U1C proteins--contain similar sequences. The five cysteine/histidine motifs found in these four proteins display amino acid similarities in addition to the cysteine and histidine residues, indicating that they participate in biological structures or functions related to the splicing process. In addition, PRP6 and PRP9 exhibit leucine repeat motifs which may be implicated in protein interactions. The prp6 and prp9 ts mutations have been mapped and sequenced.

The ability to introduce specific alterations of endogenous genes into the germ line of mice via targeted mutagenesis in embryonic stem (ES) cells has represented a major breakthrough in mouse genetics. Gene inactivation has been widely used to examine the effects of loss of function in various biological processes such as development, cellular biology, and physiology. This has already permitted the accumulation of new insights into gene function and also the creation of mouse models of human genetic diseases. Introduction of subtle mutations at specific locations of the mammalian genome is also useful to refine genetic analysis and to produce models of genetic diseases which do not necessarily result from null mutations. Several strategies have been developed, each aimed at generating subtle mutations in a given gene (6). One common limitation to all current gene-targeting procedures is the low frequency of correct targeting. This becomes a serious problem especially with use of two successive rounds of targeting, a method common to several strategies used for the generation of mutated genes devoid of foreign selection sequences. Therefore, attempts have been made to increase the efficiency of gene targeting by several means, such as increasing the size of the region of homologies with the target locus, using isogenic genomic DNA, or improving the selection procedures (6). In this report, we present an alternative approach to overcome these limitations which relies on the observation that double-strand ends of broken chromosomes are highly recombinogenic (reviewed in reference 5). Double-strand breaks (DSB) are frequently associated with DNA alteration events in eukaryotes (4, 31); during meiosis in Saccharomyces cerevisiae, for example, transient DSB are induced at a number of positions known to be hot spots for recombination (23). It has recently been shown that a unique DSB can specifically be induced in the yeast (12), plant (24), and mammalian (8, 22, 26, 28) genomes by using the yeast I-SceI meganuclease. The I-SceI protein is an endonuclease responsible for intron homing in yeast mitochondria, a process that apparently proceeds by DSB repair (18); I-SceI endonuclease can induce recombination in yeast nuclei (12). In mammalian cells, the yeast meganuclease I-SceI has been shown to efficiently induce a DSB in a chromosomal target containing an I-SceI recognition sequence. This allows DNA break repair with high frequency by recombination with a donor molecule homologous to the regions flanking the break (7, 8, 22, 25, 26, 28). We reasoned that the introduction of a DSB in an endogenous gene could increase targeting frequency at this natural locus through stimulation of the cellular recombination machinery. The gene encoding villin, a major component of the actin cytoskeleton of intestine and kidney cells (13), was chosen to develop this gene targeting strategy. We found that induction of a DSB in the target gene by using the meganuclease I-SceI resulted in greatly enhanced homologous replacement by the incoming DNA, even when the length of genomic DNA homology is reduced.