Expression vector system and a method for optimization and confirmation of DNA delivery and quantification of targeting frequency
AssigneeThe Regents of the University of California
InventorsKaarin Kerr Goncz, Dieter Cotter Gruenert, Alessia Colosimo
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Abstract
An expression vector system comprising a pair of expression vectors constructed from a wild-type and a mutant version of a reporter or selection gene, and a method for optimization and confirmation of DNA delivery and of gene targeting and for quantification of targeting frequency. Novel prokaryotic/eukaryotic DNA vectors used for DNA delivery and gene targeting assessment and targeting frequency quantification.
Claims31
An expression vector comprising a wild-type marker gene introduced into a cloning site of said expression vector.
A composition comprising a pair of expression vectors wherein one vector comprises a wild-type reporter or selectable gene and the second vector comprises a mutated-version of said wild-type reporter or selectable gene,
- said pair of vectors suitable for assessment of DNA nuclear delivery, for determination of efficiency of such DNA nuclear delivery, for optimization of said DNA nuclear delivery, for quantification of DNA targeting frequency and for gene targeting.
The composition of claim 15 wherein said wild-type reporter gene is selected from the group consisting of aminoglycoside phosphotransferase II gene (neo G418, APH), hygromycin-B-phosphotransferase gene (hygr), bleomycin resistance gene (bleo), zeocin gene (zeo), sulfonamide resistance gene (sull), hypoxantine phosphoribosyl transferase gene (HPRT), adenine phosphoribosyl transferase gene (APRT), adenosine deaminase gene (ADA), cytosine deaminase gene (CDA), dihydrofolate reductase gene (DHFR), histidinol dehydrogenase gene (hisD), puromycin-N-acetyl transferase gene (PAC), thymidine kinase gene (TK), xanthine-guanine phosphoribosyltransferase gene (gpt), diphtheria toxin gene (DT) and herpes simplex virus thymidine kinase gene (HSV-TK);
- wherein the mutated version of the wild-type selectable gene is prepared by introduction of mutation using site-directed mutagenesis or cassette insertion into said wild-type selectable gene.
A method for assessment of DNA nuclear delivery into eukaryotic cells and quantification of targeting frequency of such DNA nuclear delivery, said method comprising steps:
- a) preparing a pair of vectors comprising a wild-type and mutant version of selectable or reporter gene wherein each vector pair comprises one vector comprising a wild-type gene and the second vector comprising a mutated version of said wild-type gene;
- b) co-transfecting the cells with the vector of step (a) comprising either the wild-type or the mutant version of the selectable or reporter gene;
- c) assessing nuclear DNA delivery;
- and
- d) quantifying said nuclear DNA delivery.
Dependencies are inferred from the claim text.