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1.
Nucleic Acids Res ; 46(16): e99, 2018 09 19.
Article in English | MEDLINE | ID: mdl-29893931

ABSTRACT

Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.


Subject(s)
Bacteriophage lambda/genetics , Genetic Engineering/methods , Genetic Vectors/genetics , Integrases/genetics , Recombinant Fusion Proteins/metabolism , Transgenes/genetics , Bacteriophage lambda/enzymology , Gene Editing/methods , Gene Expression , Genetic Therapy/methods , Humans , Integrases/metabolism , Long Interspersed Nucleotide Elements/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/therapeutic use
2.
Nucleic Acids Res ; 44(6): e55, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-26673710

ABSTRACT

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, term edattH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes.


Subject(s)
Gene Transfer Techniques , Genetic Engineering/methods , Integrases/genetics , Plasmids/metabolism , Transgenes , Viral Proteins/genetics , Bacteriophage lambda/chemistry , Bacteriophage lambda/enzymology , Bacteriophage lambda/genetics , Base Sequence , Cell Line, Tumor , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression , Genes, Reporter , Genome, Human , Humans , Integrases/metabolism , Long Interspersed Nucleotide Elements , Molecular Sequence Data , Plasmids/chemistry , Viral Proteins/metabolism
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