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Targeted Chromosomal Mutagenesis Using Zinc Finger Nucleases

a technology of zinc finger nuclease and chromosomal mutagenesis, which is applied in the field of targeted chromosomal mutagenesis using zinc finger nucleases, can solve the problems of ineffective technique for introducing desired changes in the genetic material of a host cell, accompanied by problems, and technical difficulties, and achieve the effect of enhancing expression of a particular gen

Active Publication Date: 2011-01-20
UNIV OF UTAH RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene targeting—the process of gene replacement by homologous recombination or mutation—is a very useful, but typically inefficient technique for introducing desired changes in the genetic material of a host cell.
Although previously demonstrated methods of genetic transformation had been highly successful, transformation without targeted recombination has also been accompanied by problems associated with random insertion of the introduced DNA.
Additional problems associated with genetic transformation include mosaicism due to multiple integrations, and technical difficulties associated with generation of replication defective recombinant viral vectors.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Induction of Targeted Mutations

Zinc Finger Design

[0075] A pair of ZFNs were designed and constructed for a chromosomal target locus in the yellow (y) gene of Drosophila. Zinc fingers generally bind preferentially to G-rich regions of DNA, and extensive study has been performed of fingers that bind all 5′-GNN-3′ triplets (Segal et al. (1999) PNAS USA 96: 2758-2763). Because the binding sites must be in an inverted orientation with respect to each other for effective cleavage by ZFNs (Bibikova et al. (2001) Mol. Cell. Biol. 21: 289-297), the chromosomal target locus of Drosophila (y) was searched for inverted recognition sequences of the form (NNC)3 . . . (GNN)3. Such a site was identified in exon 2 with a 6-bp separation between the component 9-mer recognition sites, which is the optimal spacer for specific recognition and cleavage by ZFNs that have no added linker or spacer between the binding and cleavage domains (M. Bibikova et al. (2001) Mol. Cell. Biol. 21: 289-287). The spec...

example 2

ZFN-Induced Double Stranded Breaks Stimulate Targeted Genetic Recombination in the Presence of Homologous Donor DNA

Zinc Finger and Donor DNA Design

[0086] A pair of ZFNs were designed and constructed for a chromosomal target locus in the yellow (y) gene of Drosophila as described in Example 1.

[0087] In order to make an identifiable donor DNA for the Drosophila gene, y, the yA and yB recognition sites for the zinc fingers were replaced with two in-frame stop codons and an XhoI site. These changes were introduced by amplification with PCR primers carrying the desired sequence. Relative to the wild type y, 21 bp were deleted leaving only 3 bp of the yA recognition site, and a 9 bp replacement inserted the two in-frame stop codons and inserted the XhoI site. This mutant (yM) carries a total of 8 kb of homology to the y locus. It was inserted into a P element vector and introduced into the fly genome. The yM sequence is flanked by recognition sites for the FLP recombinase (FRT) and th...

example 3

Expression of Chimeric ZFNs in Arabidopsis in Order to Stimulate Induction of Targeted Mutations

[0099] Experimental Design The method of the present invention will be used to target and knock out the Arabidopsis TRANSPARENT TESTA GLABRA1 gene (TTG1, gene number AT5G24520 (GenBank number AJ133743). An EST for this gene has been sequenced (GenBank numbers F20055, F20056). The gene encodes a protein containing WD40 repeats (Walker et al. (1999) Plant Cell 11, 1337-1349).

[0100] Two chimeric DNA constructs will be generated consisting of (1) nucleic acid sequence encoding the promoter region from the Arabidopsis HSP18. 2 gene and (2) nucleic acid sequence encoding zinc finger proteins specific for the TTG1 gene operatively linked to a nucleic acid sequence encoding a non-specific endonuclease. The HSP18.2 promoter will confer expression in Arabidopsis and gene expression will be controlled by heat-shocking the resulting plants. The chimeric genes will be referred to as HS::ZnTTG1 A and...

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Abstract

The present invention provides for a method or methods of targeted genetic recombination or mutagenesis in a host cell or organism, and compositions useful for carrying out the method. The targeting method of the present invention exploits endogenous cellular mechanisms for homologous recombination and repair of double stranded breaks in genetic material. The present invention provides numerous improvements over previous mutagenesis methods, such advantages include that the method is generally applicable to a wide variety of organisms, the method is targeted so that the disadvantages associated with random insertion of DNA into host genetic material are eliminated, and certain embodiments require relatively little manipulation of the host genetic material for success. Additionally, it provides a method that produces organisms with specific gene modifications in a short period of time.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application is a National Phase Application of International Application No. PCT / US03 / 002012 filed on Jan. 22, 2003, which claims priority from U.S. Provisional Patent Application No. 60 / 351,035 filed Jan. 23, 2002, which is hereby incorporated herein by reference in its entirety.ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT [0002] The U.S. Government has certain rights in the invention based upon partial support by Grant R01 GM 58504.BACKGROUND OF THE INVENTION [0003] Gene targeting—the process of gene replacement by homologous recombination or mutation—is a very useful, but typically inefficient technique for introducing desired changes in the genetic material of a host cell. Only when powerful selection for the targeted product can be applied is recovery of the desired alteration possible. A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would extension of this tool ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N15/85C12N15/09A01K67/033C12N1/15C12N1/19C12N1/21C12N5/10C12N9/22C12N15/01C12N15/79C12N15/82C12N15/90
CPCA01K67/0339C12N15/902C12N15/8213C12N9/22C12N15/8257
Inventor CARROLL, DANABIBIKOVA, MARIADREWS, GARYGOLIC, KENTGOLIC, MARY
Owner UNIV OF UTAH RES FOUND
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