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T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof

A promoter and vector technology, applied in the field of genetic engineering, can solve the problems of high background of non-recombinant transformants, restriction of subcloning efficiency yield and ligation efficiency, inability to realize directional cloning, etc., and achieve the effect of improving positive cloning efficiency.

Inactive Publication Date: 2008-05-14
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has two disadvantages: first, the efficiency of subcloning is restricted by many factors such as enzyme digestion effect, target gene recovery quality, yield and ligation efficiency; second, in the process of promoter research, usually the longer the fragment , the more restriction endonuclease sites it contains, it will inevitably be limited by the selection of restriction endonuclease sites during the cloning process
However, a new problem posed by the introduction of spacer DNA is that, since circular supercoiled plasmids surge faster than linear plasmids of equivalent molecular weight in agarose gel electrophoresis, completely undigested circular supercoiled It is often mixed with the T carrier (completely cut) whose molecular weight is smaller than it, so the background of non-recombinant transformants is higher. If the pre-T carrier lacks a screening marker that is different from the T carrier, it will face more cumbersome screening work
[0010] 2. Directional cloning cannot be realized, and the screening workload is heavy
If the T vector is only used for cloning, amplification and preservation of the target gene, there is no hindrance; but if it is used for gene expression or promoter analysis, etc., more tedious screening work is required to screen out the clones in the correct direction , time-consuming and labor-intensive, especially not suitable for high-throughput screening of promoters

Method used

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  • T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof
  • T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof
  • T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Preparation of T vectors pEGFP-T, pGL3-T and pSEAP2-T

[0052] 1. Preparation of XcmI cassette

[0053] According to the sequences of plasmids pUC18 and pUC-K, the primer sequences were designed as follows:

[0054] pUC-F: 5'-CGA AGATCT CCAG CAGTTGGAGACCAAGTTTACTCA-3';

[0055] pUC-R: 5'-CAA CTCGAG CCAACCGTGCAGTGGGACAGTTACCAATGCT-3';

[0056] pUC-KF: 5'-CGA ACGCGT CCAG CAGTTGGAGACCAAGTTTACTCA-3'.

[0057] The restriction endonuclease BglII, XhoI and MluI recognition sites inserted at the 5' ends of the three primers are marked with a single line; the XcmI enzyme recognition site is marked in italics; the XcmI recognition site of the primers pUC-F and pUC-KF It still contains the first half of the rare restriction enzyme site PmeI site, which is GTTT, and the last T will constitute the 3' protruding T obtained by XcmI enzyme cleavage, marked with a wave line, and used for directional cloning analysis of promoter PCR products.

[0058] The plasmid ...

Embodiment 2

[0064] Example 2: Screening and Efficiency Detection of Forward Cloning of T Vector pEGFP-T

[0065] Taq DNA polymerase was used to amplify the minimal promoter of chemokine receptor CXCR4, and the primers were designed according to the following principles: the 5' end of the downstream primer started with "ACCC", while the upstream primer did not start with "ACCC" beginning. In this way, only when the PCR amplification product is reversely connected with the above-mentioned T vector, the recognition site of the complete endonuclease PmeI is formed (GTTTAAAC), and the pEGFP-T, pGL3-T and pSEAP2-T vectors themselves do not have this recognition site point. Therefore, reverse linking clones can be removed by digestion with PmeI. Specific steps are as follows:

[0066] CXCR4 promoter primers were designed as follows:

[0067] CXCR4-F: 5'-TACCGACCACCCGCAAACAG-3'

[0068] CXCR4-R: 5'- ACCC TAACCGCTGGTTCTCCAGA-3'

[0069] The CXCR4 PCR product was ligated with the pEGFP-T, p...

Embodiment 3

[0070] Example 3: Functional detection of recombinant T vectors

[0071] Breast cancer cells MCF-7 were cultured in RPMI 1640 medium containing 10% FBS at 37°C, 5% CO 2 cultivated under conditions. One day before transfection, take well-grown cells, trypsinize and count, and adjust the cell concentration to 2×10 5 ml, add the RPMI 1640 culture solution of the above cells in a 24-well culture plate, so that the number of cells per well is 1×10 5 . Under the mediation of Lipofectamine 2000, the above three recombinant T vectors cloned with CXCR4 promoter were transfected respectively, and pEGFP-1, pGL3-basic and pSEAP2-basic plasmids were used as negative controls. The transfection amount of each plasmid was 1 μg / well, and the amount of liposome was 2 μl per well. After culturing for 5 hours, replace with fresh culture medium, at 37°C, 5% CO 2 After 24 hours, the cells transfected with recombinant pEGFP-T / CXCR4 were observed under an inverted fluorescence microscope, and mo...

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Abstract

The invention discloses a T carrier that can directly and directionally clone a promoter and research the activity of the promoter and a construction method thereof. The construction method comprises the steps: firstly: an anterior T carrier with a Xcml box (or an AhdI box) is prepared, and the anterior T carrier comprises a DNA spacer sequence that has different resistance genes or other selective markers with the initiating carrier and two Xcml enzyme cutting site sequences that are closely connected with two sides of the DNA spacer sequence respectively; secondly, the T carrier is obtained by enzyme cutting the anterior T carrier in the first step with the Xcml enzyme or isoschizomer of the Xcml enzyme; the Xcml box in the first step is obtained by amplifying PCR, and the recognition site of the upstream primer Xcml enzyme still contains the first half part of the recognition site of the rare restriction enzyme Pmel which is used for the directional cloning analysis of the promoter PCR products. The method in the invention is also applicable to the preparation of the T carrier of a common cloning type and an expressive T carrier, and plays an important role in the genetic engineering field.

Description

technical field [0001] The invention relates to a method for constructing a vector in the field of genetic engineering, in particular to a method for constructing a T vector capable of directly cloning a promoter in a high-throughput direction and studying the function of the promoter. Background technique [0002] T vector is a linear vector used for direct cloning of PCR products, and A-T cloning technology has been proved to be extremely valuable for the cloning of PCR products. The principle of this method is to use Taq DNA polymerase to add a non-template-dependent dA base to the 3' end of the amplified product. This dA has just paired with the dT at the 3' overhang of the linear T vector for direct high-efficiency connection. without the need for restriction endonuclease digestion. [0003] In the field of promoter research, PCR amplification products of promoters often need to be cloned into vectors with reporter genes such as enhanced green fluorescent protein (EGFP...

Claims

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Application Information

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IPC IPC(8): C12N15/66
Inventor 王宝利李晓霞郭刚张镜宇梁东春左爱军孙蓓张瑞
Owner TIANJIN MEDICAL UNIV
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