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1.
Sci Rep ; 2: 438, 2012.
Article in English | MEDLINE | ID: mdl-22666541

ABSTRACT

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases.


Subject(s)
Genes, Viral/genetics , Genetic Vectors/genetics , Lentivirus/genetics , Transduction, Genetic/methods , Cell Line, Tumor , Flow Cytometry , Gene Deletion , Genetic Therapy/methods , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Lentivirus/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Virus Assembly/genetics , Red Fluorescent Protein
2.
Nucleic Acids Res ; 40(2): e14, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22110042

ABSTRACT

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.


Subject(s)
Endodeoxyribonucleases/metabolism , Gene Targeting/methods , Genes, Reporter , Cell Line , Fluorescent Dyes/analysis , Genome , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Protein Methyltransferases/analysis , Protein Methyltransferases/genetics
3.
BMC Genomics ; 12: 561, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-22085861

ABSTRACT

BACKGROUND: Mammalian olfactory receptors (ORs) are subject to a remarkable but poorly understood regime of transcriptional regulation, whereby individual olfactory neurons each express only one allele of a single member of the large OR gene family. RESULTS: We performed a rigorous search for enriched sequence motifs in the largest dataset of OR promoter regions analyzed to date. We combined measures of cross-species conservation with databases of known transcription factor binding sites and ab initio motif-finding algorithms. We found strong enrichment of binding sites for the O/E family of transcription factors and for homeodomain factors, both already known to be involved in the transcriptional control of ORs, but did not identify any novel enriched sequences. We also found that TATA-boxes are present in at least a subset of OR promoters. CONCLUSIONS: Our rigorous approach provides a template for the analysis of the regulation of large gene families and demonstrates some of the difficulties and pitfalls of such analyses. Although currently available bioinformatics methods cannot detect all transcriptional regulatory elements, our thorough analysis of OR promoters shows that in the case of this gene family, experimental approaches have probably already identified all the binding factors common to large fractions of OR promoters.


Subject(s)
Computational Biology , Conserved Sequence/genetics , Promoter Regions, Genetic , Receptors, Odorant/genetics , Algorithms , Animals , Binding Sites/genetics , Databases, Genetic , Gene Expression Regulation , Homeodomain Proteins/genetics , Mice , Sequence Alignment , Sequence Analysis, DNA , TATA Box , Transcription Factors/genetics
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