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High-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments for genetic fixed-point modification

A fragment and repeating sequence technology, applied in the field of TALE repeating fragment synthesis, can solve the problems of heavy preparatory work and troublesome preprocessing.

Active Publication Date: 2013-01-09
EDIGENE GUANGZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the disadvantages of a very large amount of preparatory work and cumbersome pre-processing (Reyon et al., 2012)

Method used

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  • High-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments for genetic fixed-point modification
  • High-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments for genetic fixed-point modification
  • High-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments for genetic fixed-point modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1 Construction of TALE-TA expression vector for upregulating human NTF-3 gene

[0126] Select the transcriptional activation binding site of the human NTF-3 gene, determine the carrier, template and primers, search for the promoter region of the human NTF-3 gene, and select the sequence (T)GGAGCCATCTGGCCGGGT as the binding site of the TALE transcriptional activator. The TALE repeat region to be synthesized can be split into six triunit fragments: GGA, GCC, ATC, TGG, CCG, GGT. The sequence structure of these fragments found in the template list (Table 4) is: G X G Z A W ,G Z C Y C W ,A W T X C Y , T X G Z G Y ,C W C Y G Z ,G Y G Z T X

[0127] According to the structure of the template and the structure of the front and back fragments, select the primers in the primer table (Table 5) as follows:

[0128] Table 6 Primer Selection Table

[0129] template

upstream primer

downstream primer

G X G Z A W

F-X5

R-W...

Embodiment 2

[0134] Example 2 Knockout of specific genes by using TALENs

[0135]1. Select the sequence of TALENs to be synthesized for a specific gene.

[0136] Human HBEGF gene is also known as DTR gene, and the gene product is the receptor of diphtheria toxin in host cells. When this gene is knocked out, the host cells should develop complete resistance to diphtheria toxin.

[0137] Search the exon sequence information of the human HBEGF gene in the NCBI (http: / / www.ncbi.nl m.nih.gov / gene / ) database, and design the TALENs used for this sequence. This region is located at positions 81-126 of the second exon of the HBEGF gene. The sequence is as follows:

[0138] CCCACTGTATCCACGgac cagctg ctaccccTAGGAGGCGGCCGGG

[0139] The uppercase letters are the TALENs binding region, and the lowercase letters are the Spacer region. The desired synthetic TALE is CCCACTGTATCCACG, CCCGGCCGCCTCCTA. The Spacer region contains a PvuII restriction site (underlined).

[0140] 2. Using ULtiMATE metho...

Embodiment 3

[0157] Example 3 Detecting the Effect of TALE-VP64 Transcription Activator with Reporter Gene

[0158] Using the method of the present invention to synthesize four TALE repeat regions whose target sites are (T)CTGGCCAAATACGTA, (T)C TGGCCTTTTACGTA, (T)CTGGCCCCCTACGTA, (T)CTGGCCGGGTACGTA, respectively, and loaded into the fusion TALE non-repeat region and VP64 The eukaryotic expression vector of transcriptional activators is in the pLenti-TALE-TA vector (see figure 2 , the vector is constructed by cloning the CMV promoter, TALE protein N-terminal non-repeating sequence, ccdB suicide gene, TALE protein C-terminal non-repeating sequence, nuclear signal peptide and VP64 transcription activator into the pLentiLox3.7 vector in sequence) .

[0159] Four reporter plasmids (see Figure 7 a). The reporter plasmids were transfected into HEK293T cells with X-tremeGENE HP transfection reagent (Roche), and culture medium containing 1 μg / ml puromycin was added for 3 days, and then normal ...

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Abstract

The invention relates to a high-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments. The high-efficiency synthesis method includes designing DNA (deoxyribonucleic acid) template and a primer for PCR (polymerase chain reaction) amplification, utilizing uracil-specific excision reagent (USER) to manufacture adhesive tail ends at two ends of the DNA segments by the uracil DNA excision method, connecting a plurality of DNA segments of a single, two or three TALE repeated units with carriers according to correct sequence to obtain recombinant carriers of TALE proteins capable of expressing specific target genes, and completing knockout, knock-in, overexpression and suppression expression of the target genes by means of the recombinant carriers. The high-efficiency synthesis method is timesaving, convenient, high in correctness and capable of realizing high throughput and meeting automatic requirements.

Description

technical field [0001] The invention relates to biogenetic engineering technology, in particular to a method for synthesizing TALE repeat fragments applied to site-directed modification of genes (especially eukaryotic genes). Background technique [0002] TALEs (Transcription activator like effectors) are the third type of secreted proteins produced by various Xanthomonas in nature to activate host gene expression or promote their own colonization (Boch and Bonas, 2010; Bogdanove et al. al., 2010; Scholze and Boch, 2011). It has been reported that the amino acid sequence of the nucleic acid binding domain of the TALEs protein has a relatively constant relationship with the nucleic acid sequence of its target site. The TALEs protein contains a highly conserved tandem repeat sequence consisting of 33-35 amino acids as units. The 12th and 13th amino acids are responsible for recognizing and binding a specific nucleotide sequence (such as NI recognizes A, HD recognizes C, NG re...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/63C12N15/85C12N15/10C12R1/64
Inventor 魏文胜杨君娇袁鹏飞
Owner EDIGENE GUANGZHOU INC
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