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Randomized libraries of zinc finger proteins

a technology of zinc finger protein and library, applied in the field of libraries of randomized zinc finger proteins, can solve the problems of limited phenotype, cumbersome method development of high throughput screening assay using these methods, and inability to use assays for transdominant genes, etc., to achieve reliable, efficient means for regulating gene expression, and regulate gene expression.

Inactive Publication Date: 2006-07-27
SANGAMO BIOSCIENCES INC
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021] The present application therefore provides for the first time methods of using libraries of randomized zinc finger proteins to screen large numbers of genes, for identifying a gene or genes associated with a selected phenotype. These libraries of randomized zinc finger DNA binding proteins have the ability to regulate gene expression with high efficiency and specificity. Because zinc finger proteins provide a reliable, efficient means for regulating gene expression, the libraries of the invention typically have no more than about 106 to about 107 members. This manageable library size means that libraries of randomized zinc finger proteins can be efficiently used in high throughput applications to quickly and reliably identify genes of interest that are associated with any given phenotype.

Problems solved by technology

However, these methods are laborious because the screening step relies heavily on conventional nucleic acid cloning and sequencing techniques.
Development of high throughput screening assays using these methods would therefore be cumbersome.
Unfortunately, this method is limited by phenotype and can only be used to assay for transdominant genes.
The main shortcoming of these methods is the inherent inefficiency of the random molecules, which vastly increases the size of the library to be screened.
For unknown gene and protein targets, the rarity of effective, bioactive peptides, antisense molecules, or ribozyme molecules imposes significant constraints on high throughput screening assays.
Furthermore, these methods can be used only for inhibition of gene expression, but not for activation of gene expression.

Method used

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  • Randomized libraries of zinc finger proteins

Examples

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example 1

Protocol for Preparation and Screening Using a Randomized Zinc Finger Protein Library Generated by Finger Grating

[0134] A. Generation of a Library Using Finger Grafting

[0135] A library of 12 different helices compatible with 5 different finger positions will be created and assembled into zinc finger proteins using a method similar to that currently used to assemble engineered 3 finger proteins (see, e.g., U.S. Ser. No. 09 / 229,037 filed Jan. 12, 1999, and U.S. Ser. No. 09 / 229,007, filed Jan. 12, 1999). Randomness will be confirmed by sequencing a representative sample. Of this library, 250,000 individual bacterial transformants will be picked and archived. The individual transformants will be combined into pools of 8 and cloned into a viral delivery vector (such as an adenoviral vector).

[0136] Viral delivery particles will be produced from each pool (there are 31,250 different pools) and tested in an appropriate assay for the identification of a desired phenotype. Assays could be ...

example 2

Protocol for Preparation and Screening Using a Randomized Zinc Finger Protein Library Generated by Codon Doping

[0154] A. Preparation of the Library Using Codon Doping

[0155] As described above, each zinc finger binds three nucleotides using four critical amino acids in the recognition helix. If each base in the codons for these amino acids was simply randomized, it would generate a library of 412 clones (1.7 million). This number is already in excess of a desired library limit of about one million to about 10 or 100 million clones and only concerns one finger (and three are to be used in these methods). However, it is not necessary to use completely random codons. Because of the redundancy of the genetic code, schemes of semi-randomization can generate representatives of all, or nearly all codons. This strategy is thus called a codon doping scheme.

[0156] One randomization scheme uses VNS instead of NNN, where N=any base, V=A or G and S=G or C. All of the codons are represented by ...

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Abstract

The present invention relates to methods of using libraries of randomized zinc finger proteins to identify genes associated with selected phenotypes.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 337,216 filed Jan. 6, 2003, which is a continuation of U.S. patent application Ser. No. 09 / 731,558 filed Dec. 6, 2000, now U.S. Pat. No. 6,503,717, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 456,100 filed Dec. 6, 1999, now abandoned; the disclosures of which are herein incorporated by reference in their entireties. [0002] This application is related to U.S. Ser. No. 09 / 229,007, filed Jan. 12, 1999, and U.S. Ser. No. 09 / 229,037, filed Jan. 12, 1999, and U.S. Ser. No. 09 / 395,448, filed Sep. 14, 1999, herein each incorporated by reference in their entirety.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0003] Not applicable. FIELD OF THE INVENTION [0004] The present invention relates to methods of using libraries of randomized zinc finger proteins to identify genes associated with selected phe...

Claims

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Application Information

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IPC IPC(8): C40B40/08C12NC12N5/00C12N5/16C12N15/10C12N15/12C12N15/63C12N15/87C12Q1/00C12Q1/68G01N33/50G01N33/53G01N33/554
CPCC12N15/1079
Inventor CASE, CASEY C.REBAR, EDWARD J.JAMIESON, ANDREW
Owner SANGAMO BIOSCIENCES INC
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