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Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus

A technology of reporter gene and lentivirus, which is applied to cells modified by introducing foreign genetic material, genetic engineering, plant gene improvement, etc., can solve the problems of no discovery, achieve method stability, overcome technical obstacles, and high transfection efficiency Effect

Inactive Publication Date: 2011-09-07
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But no reports of fluc-mrfp-ttk triple fusion reporter gene marker in iPS cell research

Method used

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  • Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus
  • Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus
  • Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Lentiviral transfection of mouse iPS cell line iPS-tet-B3

[0049] (1) Cultivation of iPS cells: The iPS-tet-B3 cell line (iPS-tet-B3 cell line (iPS-tet- For the preparation method of B3, please refer to the literature: Huang J et al. Cell Res.2009; 19(10): 1127-38) After growing to the logarithmic growth phase, passage at 1:6-9, select cells in good condition for the following virus For infection experiments, iPS cells in good condition (see figure 1 ) colonies are round or oval, with a strong sense of three-dimensionality, and individual cells cannot be distinguished;

[0050] (2) Preparation of the lentiviral vector carrying the three-fusion reporter gene fluc-mrfp-ttk: according to Entranster TM -H Transfection Reagent Operating Instructions (Engreen Biosystem Company) The pFUG-fluc-mrfp-ttk plasmid (from the laboratory of Professor Joseph C. Wu of Stanford University) was mixed with the viral packaging plasmid psPAX2 and pMD2G at a mass ratio of 3:2:1 ...

Embodiment 2

[0057] Example 2 Identification of luciferase reporter gene activity in iPS-TF cells

[0058] A. Luciferin preparation

[0059] (1) Weigh 30 mg of luciferin powder and dissolve it in 1 ml of sterile water to prepare a 200× stock solution (30 mg / ml). Mix gently by inverting until the luciferin is completely dissolved. Aliquot and freeze at -20°C for later use.

[0060] (2) Before use, quickly melt the 200× stock solution, take 10 μL and add it to 990 μL iPS cell growth medium to prepare 2× (300 ug / ml) fluorescein.

[0061] B. Cell Preparation

[0062] (1) Use 0.25% trypsin to digest iPS cells that have grown to the logarithmic growth phase and in good condition for 60 seconds, then use iPS cell growth medium to terminate the digestion reaction, and then blow the digested iPS cells with a 1000 μL pipette gun. Disperse into single cells, transfer the cell suspension to a centrifuge tube and centrifuge at 1000rpm for 5 minutes, and remove the supernatant;

[0063] (2), resusp...

Embodiment 3

[0070] Example 3 Identification experiment of iPS-TF cell pluripotency

[0071] Immunohistochemical identification of the expression of the pluripotency marker SSEA-1 was performed as follows:

[0072] (1) Fix the iPS-TF cells grown to the logarithmic growth phase in Example 1 with 4% paraformaldehyde for 15-20 minutes;

[0073] (2) Wash the fixed iPS-TF cells twice with PBS, 10 minutes each time;

[0074] (3) 4% goat serum (Sigma) blocking solution was added to block at room temperature for 30 minutes;

[0075] (4) Dilute the primary antibody (SSEA-1) to the working concentration with blocking solution (according to the antibody manual, Chemicon, USA), and incubate overnight at 4°C;

[0076] (5) Wash with PBS three times to remove unbound primary antibody, 5-10 minutes each time;

[0077] (6) Dilute the corresponding secondary antibody in PBS to the working concentration (according to the antibody instruction manual), add the secondary antibody (fluorescein isothiocyanate ...

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Abstract

The invention discloses a method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus, comprising the following steps: 1. mixing plasmids, psPAX2 and pMD2G carrying fluc-mrfp-ttk, mixing the mixed plasmids with an EntransterTM-H reagent, adding the mixed plasmids into 293T cells for mixed culture, and performing centrifugation and concentration; 2. using pancreatin to digest iPS cells and carrying out resuspension, inoculating the iPS cells to a tissue culture medium coated by Matrigel for culturing; and 3. adding concentrated virus in steps 1 to the iPS cells and culturing the virus, separating and purifying the positive iPS cells of the mrfp. The method in the invention carries out three-fusion reporter gene marking on the iPS cells, thus being capable of carrying out real-time, positioning and quantitative tracking detection on the iPS cells in animals. The method causes less damage to the iPS cells and features high transfection efficiency of more than 25%.

Description

technical field [0001] The invention belongs to the field of cell or animal labeling methods, and in particular relates to a lentivirus-mediated method for efficiently transfecting and labeling iPS cells with a triple-fusion reporter gene. Background technique [0002] The pluripotent cells that are highly similar to embryonic stem cells (Kazutoshi Takaha et al. Cell 2006, 126: 1-14) obtained from animal somatic cells through cell reprogramming are called induced pluripotent stem cells (Induced pluripotent stem cells, iPS). In the research and treatment of iPS cell transplantation, it is very important to monitor the whereabouts of the transplanted iPS cells and evaluate the therapeutic effect. At present, the monitoring of iPS cells is mainly carried out by tracer labeling technology, and the method based on reporter gene imaging better. [0003] In stem cell markers, the most commonly used reporter genes are fluorescent protein (such as green fluorescent protein GFP, red...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027C12N15/867
Inventor 王常勇刘志强王海滨段翠密林秋霞
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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