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
CN102174471AInactive Publication Date: 2011-09-07INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
Publication Date
2011-09-07
Estimated Expiration
Not applicable · inactive patent

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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%.
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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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