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Vector used for inducible expression of zinc finger nuclease (ZFN) and application thereof

A zinc finger nuclease and inducible expression technology, applied in the field of genetic engineering, can solve the problems of off-target toxicity of cells, inconvenient operation, influence of transfection efficiency, etc., and achieve the effect of simplifying the transfection process and reducing toxicity

Active Publication Date: 2014-04-30
BEIJING SINOGENE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although ZFNs have the advantage of high targeting efficiency, the current ZFNs technology system has high requirements for cell transfection efficiency
The main manifestations are as follows: 1) The main method of introducing ZFNs into cells is through transfection of ZFNs mRNA or ZFNs plasmid DNA. These methods are for transiently expressing ZFNs in cells and cannot screen transfected cells for drugs, so a large number of separation and analysis are required. It is extremely inconvenient and time-consuming to culture single-cell clones to identify on-target cells; 2) Functional ZFNs are composed of a pair of plasmids, so two plasmids or their mRNA need to be transfected at the same time, which affects the transfection efficiency. 3) If the ZFNs plasmid is stably transfected, the continuous expression of ZFNs in the cells will cause off-target toxicity to the cells

Method used

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  • Vector used for inducible expression of zinc finger nuclease (ZFN) and application thereof
  • Vector used for inducible expression of zinc finger nuclease (ZFN) and application thereof
  • Vector used for inducible expression of zinc finger nuclease (ZFN) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Construction of ZFNs-induced expression vector for sheep myostatin gene

[0048] The proteins expressed by ZFN1 and ZFN2 in the recombinant vector pTet-on-ZFN1 / 2 constructed in this example specifically recognize and bind to a specific gene site (sequence 5) of the third exon (exon3) of the sheep myostatin gene (sequence 4) ). For other genes or other sites, it is only necessary to replace the ZFN sequence that specifically recognizes the target gene or replace other gene sequences according to research needs, and the same methods and materials as those of the present invention can be used for others.

[0049] In this example, a ZFNs-inducible expression vector pTet-on-ZFN1 / 2 targeting a specific gene locus (sequence 5) of the third exon (exon3) of the sheep myostatin gene was constructed. The original plasmids used were pCI-neo plasmid, pIRESpuro3 plasmid, pTet-on-Advanced plasmid, pTRE-Tight-BI plasmid. Among them, the map of pCI-neo plasmid is as follows...

Embodiment 2

[0072] Example 2. Knocking out the myostatin gene of sheep fetal fibroblasts by inducing the expression of ZFNs

[0073] 1. Isolation and culture of primary sheep fetal fibroblasts

[0074] Cell culture medium: DMEM / F12 (product of Gibco, catalog number 12500062) + 10% (volume fraction) fetal bovine serum

[0075] Cell digestion solution: weigh 8.0g NaCl, 0.4g KCl, 1.0g glucose, NaHCO 3 , 0.35g, EDTA·tetrasodium salt 2.0g, trypsin 2.5g, with milliQ H 2 O was adjusted to 1L. Sterilize by filtration and store at -20°C for later use.

[0076] Cell freezing medium: DMEM / F12+20% (volume fraction) fetal bovine serum + 10% (volume fraction) DMSO.

[0077] 1. Isolation and culture of primary sheep fetal fibroblasts

[0078] 1. Fetal tissue collection and cell separation:

[0079] The 70-day-old Small-tailed Han sheep fetus was collected by surgery, a small piece of fetal skin tissue was cut, put in sterile saline and brought back to the laboratory, and the tissue was cut to 1mm ...

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Abstract

The invention discloses a vector used for inducible expression of ZFN and application thereof. The vector contains ColE1ori and Ampr genes and four expression cassettes. From upstream to downstream, an expression cassette 1 successively includes PCMV IE, Puror and SV40polyA, an expression cassette 2 successively includes PCMV, rtTA--Advanced and SV40polyA, an expression cassette 3 successively includes TREmod, PminCMV, a DNA fragment 1 and SV40polyA, and an expression cassette 4 successively includes TREmod, PminCMV, a DNA fragment 2 and SV40polyA. Experiments show that the invention has the following advantages: the vector provided by the invention has a gene used for drug screening, which facilitates screening and enrichment of positive cells under the condition of low transfection efficiency; the vector integrates two ZFN plasmids into one, so it only needs to transfect a single plasmid, thereby simplifying transfection process; expression time for ZFNs is controlled by using a method for inducible expression of ZFNs, so toxicity of ZFNs on cells is reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a carrier for inducing expression of zinc finger nuclease and application thereof. Background technique [0002] Gene targeting technology is a technique developed in the late 1980s to finely modify specific gene loci in higher animals. Traditional homologous recombination (HR)-mediated gene targeting methods are inefficient, only about 1 / 10 6 . Zinc finger nucleases (ZFNs) technology is a new gene targeting technology developed in recent years. ZFNs are artificially constructed hybrid molecules consisting of a polyzinc-finger domain that specifically binds to a target DNA sequence and a nuclease (Fol kI) domain that cleaves DNA molecules. The DNA binding domain enables ZFN to bind to specific DNA sequences, and the nuclease domain can cut DNA double strands. Therefore, ZFN can cut DNA in a specific DNA region, and then mutate specific gene sites by inducing homologous recombi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N9/22C12N1/15C12N1/19C12N1/21
Inventor 侯健麻富强安晓荣
Owner BEIJING SINOGENE BIOTECHNOLOGY CO LTD