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The Cre Recombinase Gene Y Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3932-3936, April 1996 Neurobiology Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene Y. WANG*, L. A. KRUSHEL*t, AND G. M. EDELMAN*t *Department of Neurobiology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037; and tThe Neurosciences Institute, 10640 John Jay Hopkins Road, San Diego, CA 92121 Contributed by G. M. Edelman, December 26, 1995 ABSTRACT Conditional gene expression and gene dele- sequences, each of which is 34 bp in length (7, 8). The tion are important experimental approaches for examining intramolecular recombination of two loxP sites (oriented ei- the functions of particular gene products in development and ther head-to-head or head-to-tail) results in an inversion or disease. The cre-loxP system from bacteriophage P1 has been deletion of the intervening DNA sequences, and an intermo- used in transgenic animals to induce site-specific DNA re- lecular recombination causes integration or reciprocal trans- combination leading to gene activation or deletion. To regulate location at the loxP site. The cre-loxP system has been tested the recombination in a spatiotemporally controlled manner, in various eukaryotic organisms and shown to function effi- we constructed a recombinant adenoviral vector, Adv/cre, that ciently in mammalian cells (9-13). It has been used in trans- contained the cre recombinase gene under regulation of the genic animals to achieve conditional gene activation or dele- herpes simplex virus thymidine kinase promoter. The efficacy tion (knockout) of targeted genes in specific tissues (5, 6, 14). and target specificity of this vector in mediating loxP- Generally, the system requires cross-breeding of two lines of dependent recombination were analyzed in mice that had been transgenic animals. One line carries an allele with the locus of genetically engineered to contain loxP sites in their genome. interest flanked at the 5' and 3' ends by loxP sequences. The After intravenous injection of the Adv/cre vector into adult other line has a cre transgene in which the expression of the cre animals, the liver and spleen showed the highest infectivity of protein is controlled by a heterologous promoter. Recombi- the adenovirus as well as the highest levels of recombination, nation between the loxP elements in the offspring is dependent whereas other tissues such as kidney, lung, and heart had upon the expression pattern of the cre transgene, which is lower levels of infection and recombination. Only trace levels defined by the specificity of the heterologous promoter (5, 6, of recombination were detected in the brain. However, when 15). Ideally, the promoter used for directing cre gene expres- the Adv/cre vector was injected directly into specific regions sion should have tissue and developmental stage specificity, or ofthe adult brain, including the cerebral cortex, hippocampus, should be inducible by a signaling molecule such as a hormone and cerebellum, recombination was detectable at the injection or cytokine (16). At present, however, such promoters are not site. Furthermore, when the Adv/cre vector was injected into generally available for each tissue or developmental stage of the forebrains of neonatal mice, the rearranged loxP locus interest, particularly for applications to specific regions of the from recombination could be detected in the injected regions central nervous system (CNS). for at least 8 weeks. Taken together, these results demonstrate An alternative approach for the delivery of transgenes into that the Adv/cre vector expressing a functional cre protein is specific target tissues involves the use of recombinant adeno- capable of mediating loxP-dependent recombination in vari- virus. This vector can infect a range of cell types with high ous tissues and the recombined gene locus may in some cases efficiency, including epithelial cells, endothelial cells, myo- be maintained for an extended period. The use of the adeno- cytes, and neurons, making it a powerful vehicle for delivery virus vector expressing cre combined with localized delivery to of foreign gene products to various tissues (17-19). After specific tissues may provide an efficient means to achieve injection of the virus into tissues, the high levels of infectivity conditional gene expression or knockout with precise spatio- and the limited spread of the virus results in a localized temporal control. expression pattern of the transgene in various somatic tissues, including the brain, liver, lung, heart, and muscle (17-19). The analysis of gene function in vivo requires the ability to In the current study, we combined the specificity of loxP- manipulate genes in a precise spatiotemporal manner. Current dependent gene recombination with the ability of adenovi- methods use germ-line transgenic techniques in which a for- ruses to effect localized gene delivery in vivo. We constructed eign gene is introduced or an endogenous gene is deleted in the a recombinant adenovirus expressing cre (Adv/cre) and dem- host genome throughout the life of the organism (1, 2). The onstrated that intravenous or intracerebral injection of the phenotypes that result from the gain or loss of function have Adv/cre vector into loxP transgenic mice results in recombi- provided insights into the physiological functions of particular nation at the loxP locus in a variety of tissues. The efficacy of genes in development and disease (3). For genes that are ex- recombination in Adv/cre-treated animals and the target pressed in multiple areas and times during development and in specificities of different delivery routes suggest a number of the adult (for example, see ref. 4), the analysis of their function useful applications for this methodology. would be facilitated by the ability to alter gene expression at restricted times and in highly localized areas. One approach to achieve such conditional gene manipulation is the use of the MATERIALS AND METHODS newly developed cre-loxP system in transgenic animals (5, 6). Construction of Adv/cre Recombinant Adenovirus. The Cre is a 38-kDa DNA recombinase that is both necessary pAdv/cre plasmid was generated using the herpes simplex and sufficient for sequence-specific recombination in bacte- virus (HSV)-cre minigene (including the HSV thymidine riophage P1. The recombination occurs between two loxP kinase gene enhancer/promoter, the cre gene coding region, The publication costs of this article were defrayed in part by page charge Abbreviations: CNS, central nervous system; HSV, herpes simplex payment. This article must therefore be hereby marked "advertisement" in virus; pfu, plaque-forming units; RT, reverse transcriptase; (3-gal, accordance with 18 U.S.C. §1734 solely to indicate this fact. ,3-galactosidase. 3932 Downloaded by guest on October 2, 2021 Neurobiology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3933 and a polyadenylylation sequence) removed from the plC-cre HSVtk Promoter Cre PolyA plasmid (a generous gift from H. Gu, National Institutes of Health) byXho I-Sal I digestion and then inserted into theXho Xhol plC-cre Sail I site of the pXCJL.1 shuttle plasmid (a generous gift from Y. Dai, Baxter Health Care, Mundelein, IL). The Adv/cre ade- Xhol pBR322 novirus was generated through homologous recombination 1iomu between cotransfected pJM17 plasmid (20) (a generous gift 4 40 from F. L. Graham, McMaster University, Hamilton, ON, Canada) and the pAdv/cre plasmid in 293 cells as previously Ad5 Ad5 described (see Fig. 1; ref. 21). The structure of the Adv/cre pAdv/Cre pJM1 7 recombinant adenovirus was confirmed both by restriction digestion of the viral DNA and by Southern blot analysis using 16 mu ORI the cre gene fragment as the labeled probe. The Adv/1-gal Ap virus used as a control is an Ela deletion recombinant adenovirus containing a 3-galactosidase (P-gal) gene with Cotransfection in 293 cells nuclear localization signal sequences driven by a human cyto- megalovirus promoter (21). The presence of the cytomegalo- virus rather than the HSV was not ex- 100 mu promoter promoter 0 1 10A d5 pected to lead to major differences in infectivity or expression. //J/ ] Both viruses were prepared as described (21). Titers of the purified viral stock were determined by measuring OD260 with Adv/cre each reading of 1.0 equivalent to 1 x 1012 plaque-forming units (pfu)/ml. tk-ce-poly Administration of the Recombinant Adenovirus. The maA- HSVtk-cre-polyA Intravenous or Stop-Tag transgenic mice (referred to as loxP mice; ref. 5) were intracerebral delivery generously provided by G. Martin (University of California, San Francisco) with permission from H. Westphal (National maAloxP Stop loxP Tag mccAloxP Tag Institutes of Health). These animals are homozygous for the F- -v WIM/ "/ /',, transgene with loxP elements flanking a 1-kb Stop sequence on a CB6 background (5; and G. Martin, personal communica- , 1536bp--- 220 bp tion; see Fig. 1). For intravenous injection, the purified ade- EcoRIEcoRI Hindlll EcoRI Hindlll novirus solution was injected through the tail veins of adult transgenic mice. For administration of the virus into the brain, maA-loxP-Stop-loxP-Tag moA-loxP-AStop-Tag adult animals were anesthetized with ketamine and xylazine. One-half microliter of Adv/cre or Adv/f3-gal solution was FIG. 1. Generation of the Adv/cre vector. The HSV thymidine infused using a stereotaxic apparatus (Stoelting) at a rate of 0.2 kinase cre minigene was excised from the pIC-cre plasmid and cloned ,Ll/min into the cerebral cortex, hippocampus, striatum, and into the shuttle plasmid pXCJ.1. As a result of homologous recombi- cerebellum on one side of the brain. For adenovirus delivery nation between pAdv/cre and pJM17 DNA, the recombinant adeno- into the neonatal mouse brain, newborn mice were anesthe- virus, Adv/cre, has the Ela region replaced by the HSV thymidine tized and on one side of the forebrain kinase-cre-poly(A) sequences.
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