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Optimized non-canonical zinc finger proteins

A technology of zinc finger protein and zinc finger, which is applied in chemical instruments and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., and can solve problems such as reduced ability

Active Publication Date: 2014-03-05
DOW AGROSCIENCES LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although zinc finger proteins containing these non-canonical fingers retain gene transcriptional regulatory functions, their ability to function as zinc finger nucleases (ZFNs) is in some cases protein decreased

Method used

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  • Optimized non-canonical zinc finger proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0311] Embodiment 1: ZFN expression vector

[0312] A ZFN comprising 4 fingers encoded as described in Examples 2 and 14 (referred to as "5 -8" and "5-9") sequences of expression vectors. Briefly, the 5-8 and 5-9 ZFNs (which comprise the nuclease domain of the type IIS restriction enzyme Fok I via a 4 amino acid ZC linker (Wah et al. (1998) Proc. Natl. Acad. Sci. USA95: The 384th-579th amino acids of the sequence of 10564-10569) were fused with four zinc finger domains) to modify the CCHC structure. Additional modifications (substitutions and insertions) were also made to the residues between the C-terminal His and Cys zinc coordination structures and / or the C-terminal residues of the C-terminal Cys of Finger 2 and / or Finger 4.

Embodiment 2

[0313] Example 2: Gene Correction of eGFP in Reporter Cell Lines

[0314] ZFNs comprising CCHC zinc fingers described herein were tested for their ability to promote homologous recombination in the GFP system described in Urnov (2005) Nature 435(7042):646-51 and U.S. Patent Publication No. 20050064474 (eg, Examples 6-11) . Briefly, 50 ng of each ZFN and 500 ng of the promoterless GFP donor (Urnov (2005) Nature) were transfected into 500,000 reporter cells using 2 μL Lipofectamine 2000 per sample according to the Invitrogen Lipofectamine 2000 protocol.

[0315] Vinblastine at a final concentration of 0.2 μM was added 24 hours after transfection and removed 72 hours after transfection.

[0316] Cells were assayed for GFP expression 5 days after transfection by measuring 40,000 cells per transfection on a Guava benchtop FACS analyzer.

[0317] Such as figure 1 As shown, most ZFNs comprising the altered CCHC zinc fingers shown in Tables 1 and 2 above promote homologous recombin...

Embodiment 3

[0318] Example 3: Editing of the chromosomal IL2Rγ gene by targeted recombination

[0319] The ZFNs described herein were also assayed in Urnov (2005) Nature 435(7042):646-51 and the endogenous IL2Rγ assay described in Example 2 of US Patent Publication No. 20050064474. Briefly, 2.5 μg of each ZFN expression construct was transfected into 500,000 K562 cells using Nucleofector (Amaxa). Genomic DNA was harvested and assayed for gene disruption at the endogenous IL2Rγ locus using the Surveyor Endonuclease Kit.

[0320] figure 2 ZFNs are shown on the upper left of . Specifically, altered zinc finger 20 refers to a CCHC zinc finger comprising the sequence HTRRCGLRGSQLV; zinc finger 21 comprises the sequence HAQRCGLRGSQLV (SEQ ID NO:53); zinc finger 43 comprises the sequence HIRTCTGSQKP (SEQ ID NO:75); zinc finger 45 comprises The sequence HIRTGCTGSQKP; zinc finger 47 comprises the sequence HIRRCTGSQKP; and zinc finger 48 comprises the sequence HIRRGCTGSQKP. Zinc fingers 20 and...

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Abstract

The invention relates to optimized non-canonical zinc finger proteins. Zinc fingers disclosed by the invention comprise CCHC zinc coordinating residues. The invention also describes zinc finger proteins, fusion proteins comprising these CCHC zinc fingers as well as polynucleotides encoding these proteins. The invention also describes methods of using these proteins for gene editing and gene regulation.

Description

[0001] This application is a divisional application of an invention application with a filing date of December 13, 2007, a Chinese application number of 200780051258.2, and an invention title of "optimized non-standard zinc finger protein". [0002] Cross References to Related Applications [0003] This application claims the benefit of U.S. Provisional Application No. 60 / 874,911, filed December 14, 2006, and U.S. Provisional Application No. 60 / 932,497, filed May 30, 2007, the disclosures of which are hereby incorporated by reference in their entirety content. field of invention [0004] The present disclosure is in the fields of genome engineering, gene targeting, targeted chromosomal integration, protein expression, and epigenome editing. Background of the invention [0005] Sequence-specific binding of proteins to DNA, RNA, proteins and other molecules is involved in many cellular processes such as, for example, transcription, replication, chromatin structure, recombinat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/11C12N15/62C12N5/10C12N15/09C12N15/82
CPCC12N15/8213C07K2319/81C12N15/8243C07K14/47C07K14/00C07K19/00C12N15/11C12N15/62C12N15/82
Inventor 其华.C.蔡杰弗里.米勒菲奥多.厄诺夫维普拉.K.舒克拉约瑟夫.F.皮托利诺莉萨.W.贝克罗比.J.加里森瑞安.C.布卢乔恩.C.米歇尔妮科尔.L.阿诺德萨拉.E.沃登
Owner DOW AGROSCIENCES LLC
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