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

Abstract

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...

Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B

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Disruption of Foxg1 expression by knock-in of Cre recombinase: Effects on the development of the mouse telencephalon

“…In primary cultures of mouse embryonic fibroblasts, in the absence of exogenous loxP sites, the introduction of the cre gene via a viral vector resulted in recombination events, growth arrest and, often, increased cell death (Loonstra et al, 2001). Similar effects have been noted in a variety of mammalian cell lines (Pfeifer et al, 2001, Silver and Livingston, 2001, Baba et al, 2005. More detailed analysis indicated that proliferating, rather than postmitotic, cells were vulnerable to the effects of Cre recombinase, as cryptic recombination events were only observed in mitotically-active cells (Loonstra et al, 2001).…”

“…These techniques, however, introduce potential confounds that need to be considered during the design of experiments and interpretation of data. For example, several in vitro studies have described effects of Cre recombinase, including growth inhibition and death, in mammalian cells lacking exogenous loxP sites (Loonstra et al, 2001, Pfeifer et al, 2001, Silver and Livingston, 2001. A recent study extended these observations to the mouse central nervous system, where it was reported that high levels of Cre recombinase in the nucleus of neural progenitors lead to impaired proliferation and increased cell death, resulting in hydrocephaly (Forni et al, 2006).…”

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…Although Cre recombinase belongs to a family of site-specific recombinases, overexpression may be cytotoxic. 45 Cremediated recombination can be monitored by expressing a selectable marker.…”