Article

Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
The Journal of Gene Medicine (Impact Factor: 1.95). 05/2006; 8(5):646-53. DOI: 10.1002/jgm.891
Source: PubMed

ABSTRACT X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-).
A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone.
Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation.
This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

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    • "The integration of foreign genes into the genome using phiC31 integrase has been reported in many organisms, including human cells (Ishikawa et al. 2006), Drosophila (Groth et al. 2004; Bischof et al. 2007; Fish et al. 2007), Mediterranean fruit flies (Schetelig et al. 2009), and mosquitoes (Nimmo et al. 2006; Labbe et al. 2011; Meredith et al. 2011). The frequency of obtaining foreign gene integration in insects varies between species. "
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    • "A site-specific integration system that inserts the transgene into a specific site of the genome would overcome these problems. Such systems have been attempted in fruit flies and other insects as well as in mammals using FLP recombinase from yeast and phiC31 integrase from phages (Horn and Handler 2005; Bischof et al. 2007; Nimmo et al. 2006; Meredith et al. 2011; Schetelig et al. 2009; Fish et al. 2007; Ishikawa et al. 2006). However, in our experiences of using FLP recombinase, we were not able to successfully establish an integration system in the silkworm, although it worked well in silkworm cell lines and embryos (Tomita et al. 1999). "
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    • "A site-specific integration system that inserts the transgene into a specific site of the genome would overcome these problems. Such systems have been attempted in fruit flies and other insects as well as in mammals using FLP recombinase from yeast and phiC31 integrase from phages (Horn and Handler 2005; Bischof et al. 2007; Nimmo et al. 2006; Meredith et al. 2011; Schetelig et al. 2009; Fish et al. 2007; Ishikawa et al. 2006). However, in our experiences of using FLP recombinase, we were not able to successfully establish an integration system in the silkworm, although it worked well in silkworm cell lines and embryos (Tomita et al. 1999). "
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    ABSTRACT: Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silk- worm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.
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