Transcriptional activation subsample effect factor nuclease and coding gene and application thereof

A transcriptional activation and encoding technology, applied in the field of genetic engineering, can solve the problems of low transfection efficiency, low efficiency, and undetectable genetic modification of targets, and achieve high targeting efficiency, high targeting specificity and high accuracy

Active Publication Date: 2016-03-02
BGI SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been applied to a variety of organisms, many targets still cannot be detected genetically modified or the efficiency is very low due to factors such as chromosomal structural inhibition of selected recognition sites, DNA modification or ineffective expression, or partial TALENs folding.
Moreover, the targeting efficiency of TALENs in cell research is largely related to the transfection efficiency of cells. Commonly used cells such as 293T have an exogenous plasmid transfection efficiency of more than 95%, while some cells have a very low transfection efficiency. For example, the mouse 3T3-L1 cell line is generally only 30% transfected, and the mutation site needs to undergo multiple enzyme digestion and enrichment methods to detect whether it has targeting activity, which brings challenges to the detection of TALENs targeting activity

Method used

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  • Transcriptional activation subsample effect factor nuclease and coding gene and application thereof
  • Transcriptional activation subsample effect factor nuclease and coding gene and application thereof
  • Transcriptional activation subsample effect factor nuclease and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The design of embodiment 1 TALENs target sequence

[0044] 1. Select the exon of the mouse chromosome 6 PPARγ2 gene (gene sequence source http: / / www.ncbi.nlm.nih.gov / ) to design TALENs target sequences.

[0045] 2. TALENs target sequences are designed according to the following main design principles:

[0046] (1) The 0th base is T, that is, the base before the first in the recognition sequence is the 0th, which is T;

[0047] (2) The length of the spacer sequence (Spacer) between the two recognition sequences is between 15-30bp;

[0048] (3) The length of the recognition sequence is between 15-24bp.

[0049] 3. Design the four sets of target sequences (i.e. RVD binding recognition sequences) shown in Table 1 according to the above principles:

[0050] Table 1

[0051]

[0052] Among them, the site sequences of the above four groups of target sequences are as follows: figure 1 shown.

Embodiment 2

[0053] The design of embodiment 2 TALENs recognition module, the construction of connection and recombinant vector

[0054] 1. According to the principle that NG recognizes T, HD recognizes C, NI recognizes A, and NN recognizes G, determine the TALENs recognition modules that respectively recognize the above target sequences, as shown in Table 2.

[0055] Table 2

[0056]

[0057]

[0058] 2. The coding sequences of the four modules NI, NG, HD and NN except the last module (the module marked with bold and underlined in Table 2) are shown in Table 3.

[0059] table 3

[0060]

[0061]

[0062] 3. The last module (the module marked with bold and underlined in Table 2) is actually half a module sequence, and its coding sequence is shown in Table 4. The rules for each module in Table 4 to recognize the corresponding bases are: LR-NI recognizes A, LR-HD recognizes C, LR-NG recognizes T, and LR-NN recognizes G.

[0063] Table 4

[0064]

[0065] 4. Perform TALEN a...

Embodiment 3

[0128] Embodiment 3 transfection and drug screening

[0129] 1. In vitro culture of 3T3-L1 cell line

[0130] (1) Select DF-12 medium with 10% FBS for culture, and add cytokine FGF if necessary;

[0131] (2) Subculture when the cell density reaches about 80%, and contact inhibition occurs when the cell density exceeds 90%;

[0132] (3) Before transfection, the cell density should be about 80%, and observed under an inverted microscope, the cells should have clear edges, clear shapes, plump and bright.

[0133] 2. The four groups of expression vector plasmids and liposomes (lipofectamine2000) listed in Table 4 were used to co-transfect 3T3-L1 cells (6-well plate).

[0134] (1) The cells were replaced with 2 ml of fresh medium (DF-12 containing 10% FBS), and the medium was preheated.

[0135] (2) In a 1.5ml EP tube, dilute about 4.0μg of each paired TALENs expression vector plasmid DNA into serum-free OPTI-MEM medium to a total volume of 50μl, and mix gently.

[0136] (3) Gent...

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PUM

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Abstract

The invention discloses transcriptional activation subsample effect factor nuclease (TALENs) and a coding gene and application thereof. The pair of TALENs are formed by fusing transcriptional activation subsample effect factors (TALEs) of specific sequences on specific recognition mouse PPARgamma2 genes and Fok1 protein, the pair of TALENs are shifted into a mouse cell system at the same time, specificity target shooting can be performed on PPARgamma2 genes of host cells, the target shooting rate is high, and the target shooting efficiency is high.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a transcription activator-like effector nuclease, its encoding gene and its application. Background technique [0002] PPAR (Peroxisome Proliferator-Activated Receptor) molecules include α, β and γ three types, distributed in different tissues, and its wide range of functions is unmatched by other biomedical molecules known today. Including metabolic syndromes such as diabetes, obesity and high blood pressure, these diseases are the main medical and health problems facing mankind in the 21st century. [0003] PPARγ2 is mainly expressed in adipose tissue and the immune system, and is closely related to adipocyte differentiation, body immunity and insulin resistance. It is the target molecule of insulin sensitizer thiazolidinediones (troglitazones, TZDs), and has become a research hotspot in recent years. . [0004] Selecting the PPARγ2 gene of the mammalian mouse 3T3...

Claims

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Application Information

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IPC IPC(8): C07K14/21C12N15/31C12N9/22C12N15/62C12N15/85C12N5/10
Inventor 王磊许奇武刘远红原辉
Owner BGI SHENZHEN CO LTD
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