Abstract
BackgroundMost common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance.Methodology/Principal FindingsWe observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected.Conclusions/SignificanceWe concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.
Highlights
Insertional mutagenesis is a modification of target DNA via incorporation of additional bases
Insertion of long DNA fragments naturally happens during retroviral infection and transposition of mobile elements. It is a byproduct of some common techniques of genetic engineering and gene therapy. It has been implicated as the cause of therapyassociated malignancies [1], and serendipitous activation of growth-promoting genes by insertion of a retroviral vector may be responsible for successful expansion of genetically engineered cells in gene therapy patients[2]
To test the prediction that successful pairing of an internal splice donor site with a host splice acceptor may result in removal of the LTR-encoded polyadenylation signal, we constructed a vector based on an HIV-1 backbone (Figure 1) that should be suitable for reversible insertional mutagenesis
Summary
Insertional mutagenesis is a modification of target DNA via incorporation of additional bases. Insertion of long DNA fragments naturally happens during retroviral infection and transposition of mobile elements. It is a byproduct of some common techniques of genetic engineering and gene therapy. Insertional mutagenesis could be used to generate pools of randomly genetically-altered cells or organisms for forward genetics applications. In this case, the mutants with a phenotype of interest are selected, and the genetic loci tagged by inserts in such mutants are further investigated as candidate regulators of the mutant phenotype. Insertional mutagenesis is a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance
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