Generation of Site-Specific Retargeting Platform Cell Lines for Drug Discovery Using phiC31 and R4 Integrases

Lieu, Pauline T.; Machleidt, Thomas; Thyagarajan, Bhaskar; Fontes, Andrew; Frey, Elizabeth A.; Fuerstenau-Sharp, Maya; Thompson, David V.; Swamilingiah, Geetha M.; Derebail, Suchitra S.; Piper, David R.; Chesnut, Jonathan D.

doi:10.1177/1087057109348941

Cited by 28 publications

(30 citation statements)

References 20 publications

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“…These results suggest that the cGPS system could address another important issue linked with random integration techniques, for unbalanced expression of 2 monomers expressed from 2 different insertion sites can have far-reaching consequences. Altogether, these results, which show that cGPS can easily match other systems based on integrases, [9][10][11][12] demonstrate that the landing pad we selected is appropriate for expression of GOIs in cellbased assays.…”

Section: Discussionmentioning

confidence: 72%

“…9,10 Recently, a dual system based on the PhiC31 and R4 integrases was used to integrate GOIs into pseudo att target sites into human cell lines, CHOS Chinese hamster ovary cells, strain S, and human embryonic stem cells. 11,12 The other option is to target foreign DNA integration via homologous gene targeting (HGT). HGT is usually inefficient in mammalian cell lines, but it is greatly stimulated if a DNA double-strand break (DSB) occurs in the vicinity of the targeted locus.…”

Section: Ell-based Assays Are Used In High-throughputmentioning

confidence: 99%

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Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

Cabaniols¹,

Ouvry

Lamamy

et al. 2010

SLAS Discovery

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The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS. (Journal of Biomolecular Screening 2010:956-967)

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Section: Discussionmentioning

confidence: 72%

Section: Ell-based Assays Are Used In High-throughputmentioning

confidence: 99%

Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

Cabaniols¹,

Ouvry

Lamamy

et al. 2010

SLAS Discovery

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“…We have previously established platform lines from a normal hESC line WA09 and an abnormal hESC line BG01V at the chromosome 13q32.3 locus by PhiC31 integrase-mediated recombination and created several reporter lines driven by an EF1a promoter [7,8,23]. Expression of reporters in these hESC lines was maintained in long-term culture at undifferentiated state.…”

Section: Construction and Retargeting Of Insulator Vectors In R4 Platmentioning

confidence: 99%

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

MacArthur

Han

Hoof

et al. 2012

Stem Cells and Development

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Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1a) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5¢ and 3¢ of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1a or CMV early enhancer/chicken b actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1a-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain-and loss-of-function experiments, and human disease modeling using hESCs.

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“…The R4 integrase as a genomic tool for integrasemediated chromosomal deletion in human cells and for generation of a site-specifi c integration platform cell lines in drug discovery was reported (Hollis et al, 2003;Lieu et al, 2009). Even though there is a high potential for R4 integrase as a useful genetic engineering tool, the molecular mechanisms of recombination mediated by R4 integrase are not well understood.…”

Section: Introductionmentioning

confidence: 99%

In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

Miura¹,

Hosaka²,

Yang³

et al. 2011

J. Gen. Appl. Microbiol.

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IntroductionIn the lysogenic phase, a temperate phage can integrate its genomic DNA into a host chromosome in a site-specifi c manner. The phage genome-encoded integrase is a site-specifi c recombinase that catalyzes the recombination between two attachment sites on the phage genome (attP) and bacterial chromosome (attB), resulting in the complete integration of the phage genome (Champbell, 1962(Champbell, , 1992. Consequently, the integrated phage DNA is sandwiched between hybrid attachment sites, attL and attR. This prophage genome can be released from the bacterial chromosome by the action of an excisionase, which is an accessory phage-encoded protein that stimulates site-specifi c recombination between attL and attR sites.There are two major families of phage-encoded sitespecifi c recombinases, based on their conserved amino acid sequences. One of them is the tyrosine recom-J. Gen. Appl. Microbiol., 57, 45 57 (2011) The site-specifi c integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specifi c recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for effi cient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His 6 )-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purifi ed protein preparation retains the site-specifi c recombination activity which catalyzes the site-specifi c recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for effi cient in vitro intermolecular recombination. In addition, a gel shift assay showed that His 6 -GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifi cally. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specifi c recombination by the R4 integrase have been defi ned more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.

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Generation of Site-Specific Retargeting Platform Cell Lines for Drug Discovery Using phiC31 and R4 Integrases

Cited by 28 publications

References 20 publications

Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

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