Review
Site-specific gene targeting for gene expression in eukaryotes

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Abstract

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase–DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.

Introduction

Sustainable gene expression, through genomic modification, is a long-desired goal for DNA transfer systems. The low frequency of integration into active regions of chromosomes and the concomitant lack of sustainable gene expression, remain major problems with many gene expression paradigms. Gene expression is often curtailed when integration into inactive regions of the chromosome occurs. New methods for site-directed targeting that allow persistent gene expression are needed for many of the new DNA-based therapeutics, such as cell therapy, tissue engineering and gene therapy, to be safe and efficient. Furthermore, transgenic organisms, which are widely used to develop disease models, produce recombinant protein [1], [2] and will soon provide a source of organs for xenotransplantation, are also hampered by a lack of chromosomal targeting methods. Site-specific targeting that enables continued gene expression, following delivery, is critical for the ‘new biology’ approaches to fulfill their promise, in both discovery research and applied endeavors.

There are several current methods for achieving transgene integration: firstly, random insertion into chromosomes, which has a frequency of one event in 10,000; secondly, homologous recombination, which is very specific but has a frequency of one in 1,000,000; thirdly, retroviruses, which integrate but randomly; and finally, adeno-associated viruses (AAVs) vectors, which lose the site-specific integration ability. Advances are being made in all of these fields to increase the frequency of legitimate site-directed gene targeting. Efforts to increase the frequency of homologous recombination are underway [3] and one of the more promising approaches is the use of double strand breaks [[4], [5•]]. Additionally, much effort has been made to design viral vectors that facilitate site-specific gene targeting [6], [7], [8], [9], [10], [11], [12], [13]. There is, however, still a need for a convenient method for permanently modifying chromosomes at specific sites.

Site-specific recombinases (see [14], [15] for reviews on recombinase enzymes) hold promise for providing novel approaches to increase the efficiency of targeted integration. Site-specific recombinases catalyze the introduction or excision of DNA fragments (Fig. 1). The crystal structure and topology of four members of this family of enzymes (Cre, XerD, HP1 and Flp) have been shown to have similar three-dimensional structures and conserved catalytic regions [14], [16], [17]. These enzymes recognize a relatively short, unique nucleic acid sequence, which serves for both recognition and recombination. The fundamental mode of action for each of these enzymes is quite similar. Each recombination site is composed of short inverted repeats (6, 7 or 8 base pairs [bp] in length) and the length of the DNA-binding element is also quite consistent (11–13bp in length) [14], [15], [17] (Fig. 2).

One of the most widely studied site-specific recombinases is the enzyme Cre, from the bacteriophage P1. Cre recombines DNA at a 34bp sequence called loxP, which consists of two 13bp palindromic sequences flanking an 8 bp core sequence. In this manuscript, we will explore technological advances made with this system during the past year. These advances include innovative modifications of the Cre-lox systems and the discovery of long-predicted Cre-like sites in the mammalian genome.

Section snippets

Cre in eukaryotic cells

Cre can direct site-specific integration of transgenes into the genome of eukaryotic cells. Recently, it has been shown that Cre contains amino acid sequences that direct translocation of the protein into the eukaryotic nucleus; this transport is necessary for this bacteriophage enzyme to function in diverse eukaryotic systems [18radical dotradical dot].

Most current methods using recombinases in eukaryotic systems require modification of the host genome first to include recognition sequences. Targeted gene insertion

Manipulations of Cre/loxP gene expression cassettes in transgenic animals

As mentioned, a frequently encountered problem in transgenic animal studies is the lack of expression of the transgenes as a result of either insertion into transcriptionally inactive chromatin, or inadequate transcription from uncharacterized promoters. Even when both Cre and the reporter gene cassettes are transcribed, the latter may lie in a chromatin configuration inaccessible to recombination by functional Cre, thus confounding analyses.

Several papers address these problems. Because the

Inducible and tissue-specific Cre expression

Several investigators have reported ways of making in vivo Cre expression both inducible and tissue specific. Inducibility of tissue-specific Cre expression allows one to perform genetic manipulations that could be otherwise lethal, produce developmental abnormalities, compensatory changes, or pleiotropic effects, and that can be studied against the uninduced background of the same animal. Toward this end, two approaches have been employed in vivo. In the first approach, inducibility is

Use of Cre/loxP in gene therapy for cancer

Many strategies for treatment of cancer have involved delivery of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by gancyclovir administration, which inhibits tumor growth in the presence of thymidine kinase. In order to reduce the toxicity of such a therapy, it is desirable to limit expression of the thymidine kinase to the tumor by expressing it under a tumor-specific promoter. Unfortunately transcription from tumor-specific promoters, such as the carcinoembryonic antigen

Further possibilities for chromosomal targeting

Other members of the recombinase enzymes, though prokaryotic in origin, have also been shown to function in eukaryotic systems [[18••], [19•], [20], [21••], [22••]]. Recently, the eukaryotic type 1B topoisomerases have been shown to contain regions that are highly homologous to the catalytic region of these site-specific recombinases [33]. This observation may lead to the development of technology that uses eukaryotic enzymes, such as topoisomerases, to augment specific integration.

Previous

Conclusion

Site-specific recombinases are increasingly useful reagents for genomic manipulations that allow precise, spatially and temporally controlled changes in gene expression. In novel uses of the recombinase Cre in transgenic animal models, genes can be engineered for expression even when known promoter regions have not yet been identified. Advances in the development of disease modeling have also been made using ever more specific targeting, both spatially and temporally, of specific genes. Both

Update

Since submission of this manuscript, additional works, detailing new approaches to achieving site-specific integration in eukaryotes, were published. Dutheil et al. [35radical dot] and Tsunoda et al. [36] discuss new data that increase the understanding of site-specific integration of AAV. Both groups study the chromosomal sequences and mechanisms necessary for AAV-specific insertion into the human chromosome. Although several questions still remain with respect to the requirements for targeted

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • radical dot of special interest

  • radical dotradical dot of outstanding interest

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