Method for enhancing nonconformable gene expression in human cells

A technology for human embryonic kidney cells and cells, which can be used in DNA/RNA fragments, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., and can solve problems such as low efficiency

Active Publication Date: 2014-07-16
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Episomal plasmids also have certain disadvantages, s

Method used

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  • Method for enhancing nonconformable gene expression in human cells
  • Method for enhancing nonconformable gene expression in human cells
  • Method for enhancing nonconformable gene expression in human cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] The preparation of embodiment 1, EBNA-D500mRNA

[0121] 1. Preparation of templates for in vitro transcription

[0122] The pRN3P-EBNA-D500 vector was linearized with restriction endonuclease SfiI, and the digested product was purified with a DNA purification and recovery kit, and used as a template for in vitro transcription of EBNA-D500, which can be stored at -80°C for future use.

[0123] 2. In vitro transcription of EBNA-D500mRNA

[0124] Utilize mMESSAGE In the T3 mRNA in vitro transcription kit, each component was added as shown in Table 1, reacted at 37° C. for 2 hours, and performed an in vitro transcription reaction to obtain EBNA-D500 mRNA (SEQ ID No.2).

[0125] Table 1 In vitro transcription system

[0126]

[0127] 3. Add 1 μL TURBO DNase to the in vitro transcription system, react at 37°C for 20 minutes, and digest the DNA template.

[0128] 4. Using Microspin TM S-200HR column (purchased from GE, Cat. No. 27-5120-01), the mRNA product obtained i...

Embodiment 2

[0135] Example 2. Chemical co-transfection of EBNA-D500mRNA and Episomal plasmid in human embryonic kidney cell line 293FT

[0136] The Chinese name of Episomal plasmid is "non-integrated episomal plasmid", that is, a plasmid containing OriP / EBNA sequence, wherein OriP is in cis structure and EBNA is in trans structure, and can be used as an expression vector for any mammalian cell.

[0137] Episomal plasmid and EBNA-D500 mutant system such as figure 1 shown.

[0138] pCEP4-EGFP and pCXLE-EGFP are two different Episomal plasmids.

[0139] Specific steps are as follows:

[0140] 1. Passage human embryonic kidney cells 293FT to a 24-well plate one day before transfection, and the cell confluence is 40-50%.

[0141] 2. Replace the fresh 293FT cell culture medium two hours before transfection.

[0142] 3. According to Lipofectamine TM 2000 instructions for transfection. The mass ratio of liposomes to plasmids was 3:1. In the experimental group, 0.5μg EBNA-D500mRNA and 0.8μg...

Embodiment 3

[0153] Example 3, Electrotransfection of EBNA-D500mRNA and Episomal plasmids in human fibroblasts

[0154] 1. Subculture human fibroblasts the day before transfection.

[0155] 2. Using Invitrogen Electrotransfection instrument, set the electrotransfection parameters before transfection, the parameters are 1550 volts, 10 milliseconds, 3 times.

[0156] 3. Dissociate human fibroblasts into single cells with 0.25% trypsin / EDTA, centrifuge to pellet the cells, discard the culture medium, resuspend the cells in PBS, and calculate the total number of cells.

[0157] 4. Add an appropriate volume of electrotransfer fluid Buffer R (Neon TM Electroporation kit comes with) to resuspend cells, adjust the cell concentration to 1x107 / mL, add 1 μg EBNA-D500mRNA and 0.5 μg Episomal plasmid (pCEP4-EGFP) to 10 μL of cells or add 0.5 μg Episomal plasmid (pCEP4-EGFP ), and the group without any plasmid was used as the blank control group.

[0158] Five, according to Neon TM Instructions fo...

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Abstract

The invention discloses a method for enhancing nonconformable gene expression in human cells. The invention discloses an EBNA-D500DNA molecule, the sequence of which is shown as SEQ IN NO.1. The method disclosed by the invention has the advantage of overcoming the defect that the Episona1 plasmid has lower in human cell line without influencing the unconformability and the characteristic of long-time expression. Moreover, the EBNA-D500DNA is capable of remarkably improving the efficiency of having the Episona1 plasmid to carry out somatic cell reprogramming, can be used for producing human induced pluripotent stem cells which are free of exogenous genes and high in safety, and is applicable to regenerative medicine.

Description

technical field [0001] The present invention relates to a method of enhancing expression of non-integrated genes in human cells. Background technique [0002] Stem cells and regenerative medicine are important research areas of biomedicine in recent years, and have great clinical application value. Pluripotent stem cells can be differentiated into functional cells of various tissues and organs, which can be used to study human development, make disease models, and replace damaged and diseased cells through cell transplantation to promote wound repair and treat diseases. Stem cells and regenerative medicine will change the traditional treatment methods for diseases such as necrosis and injury, and bring about revolutionary changes in the mechanism research and clinical application of diseases. How to ensure and improve the safety of stem cells is a key issue in regenerative medicine. Conventional methods of exogenous gene expression have several drawbacks. Stem cell differ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/67
Inventor 那洁段福宇李津旸
Owner TSINGHUA UNIV
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