Exogenous-gene removable slow virus controlled expression carrier system and application

A technology for expressing vectors and exogenous genes, applied in the field of genetic engineering, can solve the problems of decreased expression of exogenous genes, causing host immune response, non-integration of exogenous genes, etc., to achieve the effect of avoiding interference

Inactive Publication Date: 2013-10-16
JIANGSU INST OF NUCLEAR MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In various viral expression vector systems: Retrovirus vectors can usually only transfect actively dividing cells, and have the ability to insert mutations or replicate next-generation viruses, which has certain risks; Adenovirus vectors can To ensure the high-efficiency expression of foreign genes, but its immunogenic protein may also be expressed, causing host immune response, in addition, the foreign gene it carries is not integrated into the cell chromosome, so the foreign gene in the dividing progeny cells The expression level of the gene continues to decline; while the lentiviral vector can be stably integrated into the chromosome of the host cell and can be expressed continuously for a long time in dividing and non-dividing cells. Therefore, the lentiviral vector system has become one of the most widely used expression vectors at present. one

Method used

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  • Exogenous-gene removable slow virus controlled expression carrier system and application
  • Exogenous-gene removable slow virus controlled expression carrier system and application
  • Exogenous-gene removable slow virus controlled expression carrier system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning of embodiment 1 Tet-response element (TRE) and the most simplified CMV promoter sequence (TREminiCMV)

[0043] Use a plasmid extraction kit to extract the plasmid in JM109 / pTRE-Tight, use it as a template, and obtain the TREminiCMV sequence by PCR amplification. The primers used are as follows:

[0044] TREminiCMV-For: 5’-GAG GG G ATC CC T TTC GTC TTC ACT C-3' (the primer sequence is shown in SEQ ID NO: 1), the underlined part is the BamH I restriction endonuclease recognition site;

[0045] TREminiCMV-Rev: 5'-CGA GAT ATC GAA TTC TCC AGG CGA TC-3' (the primer sequence is shown in SEQ ID NO: 2), the underlined part is the EcoR V restriction endonuclease recognition site;

[0046] The PCR reaction system is: pTRE-Tight1μl, 10μmol / L upstream and downstream primers 1μl each, 2.5mmol / L dNTP4μl, 10×Pyrobest Buffer 5μl, 5U / μl Pyrobest DNA polymerase 0.5μl, add double distilled water to make up 50 μl.

[0047] The conditions of the PCR reaction were: pre-denatura...

Embodiment 2

[0050] Example 2 Cloning of green fluorescent protein (EGFP) cDNA

[0051] Using the pBMN-GFP plasmid as a template, EGFP cDNA was amplified by PCR, and the primers used were as follows:

[0052] EGFP-For: 5'-CGA GAT ATC ATG GTG AGC AAG GGC GAG-3' (the primer sequence is shown in SEQ ID NO: 3), the underline is the EcoR V restriction endonuclease recognition site;

[0053] EGFP-Rev: 5'-CGC GAC CCT TGG TC T TAC TTG TAC AGC TCG-3' (the primer sequence is shown in SEQ ID NO: 4), the underline is the Ahd I restriction endonuclease recognition site;

[0054] The PCR reaction system is: pBMN-GFP1μl, 10μmol / L upstream and downstream primers 1μl each, 2.5mmol / L dNTP4μl, 10×PCR Buffer5μl, 5U / μl rTaq DNA polymerase 0.5μl, add double distilled water to make up 50μl .

[0055] The conditions of the PCR reaction were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and 35 cycles; final ext...

Embodiment 3

[0058] Mutation of embodiment 3 LTRU3 region and insertion of loxp sequence

[0059] The site-directed mutation of ACCGGT in the 3'LTR U3 region of the pSMPUW vector, the mutated sequence is ACGCGT, and the Mlu I restriction site is introduced. The vector plasmid mutation primers are as follows:

[0060] Mut-For: 5'-TTG CTT TAT T TG TAA ACG CGT GCA GCT GCT-3' (the primer sequence is shown in SEQ ID NO:5);

[0061] Mut-Rev: 5'-TAG CAT CAC AAA TTT CAC AAA TAA ATT CCG-3' (the primer sequence is shown in SEQ ID NO:6);

[0062] The reaction system is: 0.5 μl of pSMPUW plasmid, 1 μl of 10 μmol / L upstream and downstream primers, 4 μl of 2.5 mmol / L dNTP, 5 μl of 10×Pyrobest Buffer, 0.5 μl of 5 U / μl Pyrobest DNA polymerase, and 50 μl of double distilled water .

[0063] The conditions of the PCR reaction were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 7 minutes and 30 seconds, and 35 cycles; fi...

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Abstract

The invention discloses an exogenous-gene removable slow virus controlled expression carrier system and an application. The system comprises a reaction/regulation plasmid and a packaging plasmid; a construction method is as follows: a loxP sequence is inserted into a pSMPUW 3'LTR through site-specific mutagenesis, an original promoter is replaced by a TRE and a simplest CMV promoter, and an exogenous reporter gene is inserted into an MCS to obtain the reaction plasmid; a CMV-rtTA2S-M2 sequence, an EMCV sequence and a Cre-ER sequence are inserted into the pSMPUW subjected to site-specific mutagenesis to obtain the regulation plasmid. The application is as follows: packaging is conducted to obtain a reaction/regulation slow virus; both the reaction slow virus and the regulation slow virus are cotransfected to a target stem cell, a Dox induction exogenous reporter gene is used for expressing and tracing the image, and finally a 4-OH-tamoxifen is used for inducing and excising the exogenous reporter gene. According to the invention, the exogenous reporter gene is expressed in a controlled manner in vivo and can also be removed immediately to avoid disturbing the normal physiological function of a target cell.

Description

technical field [0001] The present invention relates to a lentiviral expression vector system carrying an exogenous reporter gene, in particular to a lentiviral controlled expression vector system whose expression is regulated by doxycycline (Dox) and the exogenous reporter gene carried can be removed from the host chromosome , and the application of this system in stem cell tracking technology. It belongs to the technical field of genetic engineering. Background technique [0002] Overexpression of exogenous reporter genes requires a continuous, stable and efficient gene transfer and expression system. At present, the expression of most foreign genes is accomplished by virus expression vector system. In various viral expression vector systems: Retrovirus vectors can usually only transfect actively dividing cells, and have the ability to insert mutations or replicate next-generation viruses, which has certain risks; Adenovirus vectors can To ensure the high-efficiency exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867G01N21/64G01N15/14
Inventor 付强杨润林邓黎莉彭莹丁月娣范俊
Owner JIANGSU INST OF NUCLEAR MEDICINE
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