pSEPT Plasmid
Report of Invention:
4/29/2010
C11082: High Efficiency Human Gene Targeting with Recombinant AAV and a Synthetic Exon Promoter Trap (SEPT) - pSept PlasmidValue Proposition:
• SEPT targeting approach is generally useful in a variety of human cell lines
• Simple method, eliminating need for customizing selectable marker for each gene targeted
• The combined use of rAAV and the SEPT approach can significantly improve the efficiency of gene targeting in human cells.
• Method does not require the disruption of an endogenous exon and can therefore be used for the generation of human gene knockin cell lines Technical Details: BACKGROUND
Gene targeting via homologous recombination is the most definitive means of assessing gene function. While routinely applied to diverse organisms such as bacteria, yeast and rodents, established methods of gene targeting have been significantly less efficient when applied to human cell lines. The low rate at which homologous recombinants can be recovered from human cell populations seems to be due to a lower rate at which exogenous DNA can be introduced into cells and integrated into the genome, and possibly to a lower rate of homologous recombination as well. A major advance in the targeting of human genes was the development of the promoter-trap targeting which have been successfully employed in the targeting of many human loci and are generally considered the method of choice for plasmid-based gene targeting. Although, the method of choice this technique has its own limitations which creates a need for improved versatility for the creation of knockout or knockin cell lines for any transcriptionally active human gene.
TECHNOLOGY
JHU Researchers have developed a novel vector configuration termed the synthetic exon promoter trap (SEPT). The plasmid pSEPT contains the SEPT cassette flanked by tandem LoxP sites oriented so that transient expression of the Cre recombinase results in excision of all sequences between them. This targeting element has further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. This versatile method was used to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.
Looking for Partners: The pSEPT technology can be used to study the role of many genes in cancer biology, providing excellent reagents for drug discovery and target validation.
Publications/Associated Cases: 1. Rago, C., Vogelstein, B., & Bunz, F. (2007). Genetic knockouts and knockins in human somatic cells. Nature Protocols, 2(11), 2734-2746.
2. Topaloglu, O., Hurley, P. J., Yildirim, O., Civin, C. I., & Bunz, F. (2005). Improved methods for the generation of human gene knockout and knockin cell lines. Nucleic Acids Research, 33(18), 1-7.
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