Bacteriophage P1 site-specific recombination

540

473

The use of Cre͞loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER T show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.genotoxicity ͉ conditional knockout

mentioning

confidence: 99%

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells

Loonstra

Vooijs²,

Beverloo³

et al. 2001

540

473

“…The Cre͞loxP system is a phage P1-derived, site-specific recombination system in which Cre recombinase catalyzes the recombination between 34-bp loxP recognition sequences (8)(9)(10). The loxP sites are engineered around crucial exons by gene targeting in such a way so as not to affect normal gene function (11).…”

mentioning

confidence: 99%

Adeno-associated virus effectively mediates conditional gene modification in the brain

Kaspar

Vissel²,

Bengoechea³

et al. 2002

182

148

The Cre͞loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial-and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein͞Cre recombinase (GFP͞Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates ␤-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP͞Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.gene transfer ͉ Cre recombinase ͉ transgenic mouse ͉ loxP ͉ ROSA26

“…catalyzes the recombination of two 34-bp sequences called loxP, which consist of two 13-bp palindromic sequences flanking an 8-bp core sequence (1,2). Because of the high efficiency of the Cre͞loxP system in mammalian cells (3,4), this system is now widely used for genetic engineering in vitro and in vivo (5,6).…”

mentioning

confidence: 99%

Delivery of the Cre recombinase by a self-deleting lentiviral vector: Efficient gene targeting in vivo

Pfeifer

Brandon²,

Kootstra³

et al. 2001

221

201

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G2͞M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Credependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.gene transfer ͉ regulated gene expression ͉ Cre toxicity T he site-specific recombinase of the bacteriophage P1, Cre, catalyzes the recombination of two 34-bp sequences called loxP, which consist of two 13-bp palindromic sequences flanking an 8-bp core sequence (1, 2). Because of the high efficiency of the Cre͞loxP system in mammalian cells (3, 4), this system is now widely used for genetic engineering in vitro and in vivo (5, 6). Transfer of Cre in vivo can be achieved by the use of transgenic mice or by viral delivery to the target organ(s). Thus far, the transfer of Cre by using nonviral delivery vehicles such as liposomes has only been described for use in vitro (7).We have generated lentiviral vectors that carry the Cre recombinase and have analyzed their ability to mediate loxPspecific recombination. Lentiviruses are enveloped RNA viruses that belong to the family of complex retroviruses (for review see refs. 8 and 9). In contrast to prototypic retroviruses, such as murine leukemia virus, lentiviruses are able to transduce both dividing and nondividing cells. Lentiviral vectors pseudotyped with the G protein of the vesicular stomatitis virus can transduce a wide range of cells and have great potential for human gene therapy (10-13).The Cre lentiviral vectors efficiently transferred Cre to the target cells and induced Cre-mediated recombination of loxP sites in vitro and in vivo. The Cre-expressing cells unexpectedly exhibited a reduction in proliferation and accumulated in the G 2 ͞M phase of the cell cycle. To circumvent this toxic effect of Cre, we designed a Cre lentivirus vector that is itself subject to Cre-mediated excision (self-deleting or LV-Cre-SD). In this way, Cre expression is limited to only the time necessary for recombination. Our studies show that this approach works efficiently in vitro and in vivo. Materials and MethodsAnimals. Homozygous R26R mice (12-20 weeks of age) were anesthetized by i.p. injection of a ketamine (150 mg/kg)͞xylazine (15 mg/kg) mixture. Virus (3 l) was injected either ster...