Expanding Flp-RMCE options: the potential of Recombinase Mediated Twin-Site Targeting (RMTT)

Turan, Soeren; Jian, Qiao; Madden, Sally; Benham, Craig J.; Kotz, Marina; Schambach, Axel; Bode, Jürgen

doi:10.1016/j.gene.2014.06.002

Cited by 8 publications

(6 citation statements)

References 58 publications

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“…Retroviral or lentiviral screens can be coupled to recombinase-mediated cassette exchange strategies to allow researchers to "re-use" individual integration sites in order to assess different expression cassettes harboring a variety of cis-regulatory elements, both constitutive and tissue-specific, with relative ease. 53,54 Human pluripotent stem cells (hPSCs) offer an ideal platform for such screens and can streamline the discovery and the validation phases, as discussed further below.…”

Section: Discovery Of Gshsmentioning

confidence: 99%

Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy

2016

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Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy.

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Section: Discovery Of Gshsmentioning

confidence: 99%

Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy

2016

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“…Another recent improvement of RMCE is the implementation into lentviral vectors for potential gene therapy applications , and the combination with other genome-editing technologies, such as TALENs and CRISPR/Cas (see also 5.8).…”

Section: Applied Use Of Tyrosine Recombinasesmentioning

confidence: 99%

Cre Recombinase and Other Tyrosine Recombinases

et al. 2016

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Tyrosine-type site-specific recombinases (T-SSRs) have opened new avenues for the predictable modification of genomes as they enable precise genome editing in heterologous hosts. These enzymes are ubiquitous in eubacteria, prevalent in archaea and temperate phages, present in certain yeast strains, but barely found in higher eukaryotes. As tools they find increasing use for the generation and systematic modification of genomes in a plethora of organisms. If applied in host organisms, they enable precise DNA cleavage and ligation without the gain or loss of nucleotides. Criteria directing the choice of the most appropriate T-SSR system for genetic engineering include that, whenever possible, the recombinase should act independent of cofactors and that the target sequences should be long enough to be unique in a given genome. This review is focused on recent advancements in our mechanistic understanding of simple T-SSRs and their application in developmental and synthetic biology, as well as in biomedical research.

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“…1 a). In the literature, a limited number of multiplex cell lines combining different RMCE systems are described (Kameyama et al 2010 ; Turan et al 2010 , 2014 ; Anderson et al 2012 ), however, none couples the advantages of multiplexing to the tailored production of viral vectors. In this work, the transgene expression flexibility reported and validated for 293 FLEX (Coroadinha et al 2006c ) was extended with the flexibility of viral envelope(s) expression to produce tailor-made recombinant retrovirus.…”

Section: Discussionmentioning

confidence: 99%

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange

et al. 2018

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ObjectiveDevelop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.ResultsDual-FLEX cells (dFLEX) contain two independent recombinase-mediated cassette exchange (RMCE) systems which confer flexibility to the expression of different transgene and envelope combinations. The flexible envelope expression in dFLEX cells was validated by pseudotyping retrovirus particles with three different viral envelope proteins—GaLV, 4070A and VSV-G. Our results show that dFLEX cells are able to provide high titers of infectious retroviral particles with a single-copy integration of the envelope constructs after RMCE. The integrated CRE/Lox tagging cassette was amenable to express envelope proteins both using constitutive (i.e. CMV) and inducible (i.e. Tet-on) promoters.ConclusionsdFLEX cell line provides predictable productivities of recombinant retrovirus pseudotyped with different envelope proteins broadening the tropism of particles that can be generated and thus accelerating the research and development of retrovirus-based products.Electronic supplementary materialThe online version of this article (10.1007/s10529-018-2515-6) contains supplementary material, which is available to authorized users.

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Expanding Flp-RMCE options: the potential of Recombinase Mediated Twin-Site Targeting (RMTT)

Cited by 8 publications

References 58 publications

Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy

Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy

Cre Recombinase and Other Tyrosine Recombinases

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange

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