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Rat induced multipotent stem cells and preparation method thereof

A technology for pluripotent stem cells and stem cells, which is applied in the field of induced pluripotent stem cells and their preparation, and can solve the problems of inability to explore germline embedding, failure to maintain ES-like cells for a long time, and failure to produce teratoma.

Inactive Publication Date: 2010-06-23
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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Problems solved by technology

However, the scientific community has not been able to successfully establish a rat embryonic stem cell (ES) line for decades. It was first reported in 1994 that P. Lannaccone's laboratory announced that it had obtained rat ES cells and made a chimera, but three years later, they However, the results of the year were overturned because of the contamination of mouse embryonic stem cells (Iannaccone, P.M., Taborn, G.U., Garton, R.L., et al. (1994). Dev.Biol.163, 288-292; Brenin, D., et al.(1997).Transplant.Proc.29, 1761-1765); at the beginning of this century, several laboratories successively reported the establishment of cell lines similar to rat embryonic stem cells (ES-like) (Demers, et al. .(2007), Cloning and stem cells 9, 512-522; Vassilieva, S., et al.(2000), Exp.Cell Res.258, 361-373; Notarianni, E., et al.(2001), Placenta 22, 111-123; Fandrich, F., et al. (2002), Nat. Med. 8, 171-178; Buehr, M., et al. (2003), Biol. Reprod.68, 222-229 ; Mullins, L.J., et al.(2004), J.Physiol.554, 4-12; Tesson, L., et al.(2005), Transgenic Res.14, 531-546), but the defect is: ES- Like cells cannot be maintained for a long time; the experimental results of verifying the pluripotency of ES-like cells in vivo are not ideal, that is, no teratomas can be produced; there are no reports of successful chimera rats, and ES-like cells injected into blastocysts Cells can only differentiate into extraembryonic tissues, so germline insertion cannot be explored; moreover, identification of markers characteristic of stem cells is limited
The establishment of rat embryonic stem cells has become a huge limiting factor that hinders the wide application of rats in biomedicine. According to these previous research results, the use of traditional methods (the establishment method of mouse ES cells) to establish rat ES cells is quite difficult

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  • Rat induced multipotent stem cells and preparation method thereof
  • Rat induced multipotent stem cells and preparation method thereof
  • Rat induced multipotent stem cells and preparation method thereof

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Experimental program
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Embodiment 1

[0045] Embodiment 1, Construction of lentiviral vector

[0046] 1.1. Query the coding regions of specific genes (Oct4, Sox2, c-Myc, Klf4, Lin28, Nanog) specifically expressed or highly expressed in stem cells from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) , design primers according to the sequence of the coding region, and introduce restriction sites, the primer sequences are shown in Table 1 (wherein F represents the forward primer, R represents the reverse primer).

[0047] Table 1

[0048]

[0049] Note: The uppercase letters in the primer sequences are the restriction restriction sites introduced.

[0050] 1.2. PCR amplification

[0051] Using the total human cDNA as a template, PCR amplification was performed using the primers of each gene in Table 1, as follows:

[0052] Reaction system (25 μl): 2.5 μl of 10×pfx Mix, 0.2 μl of AccuPrime pfx enzyme, 0.25 μl of upstream and downstream primers (50 μM), 0.25 μl of template, and 21.55 μl of ddH2O.

[0053] Reac...

Embodiment 2

[0057] Embodiment 2, cell culture

[0058] 2.1. Culture of primary rat bone marrow cells (BMC)

[0059] Take the femur and tibia of male SD rats at 6 weeks, soak them in 75% ethanol for 10 minutes, wash them three times with PBS containing double antibodies (penicillin, streptomycin), cut the two ends of the bones, and wash with 10% fetal bovine serum (FBS) α-MEM medium sterile syringe into the bone marrow into a 15mL sterile centrifuge tube, centrifuge at 1200rpm for 5min, use the above medium to resuspend the cells once, centrifuge again at 1200rpm for 5min, resuspend the medium again and transfer the cells into in a culture bottle. Three days later, the medium was changed, and micro-clones were seen. About a week later, the ratio of 1 to 3 was passed. The cells were washed twice with PBS and digested with 0.25% trypsin at 37°C for 5 minutes. Can keep in good condition.

[0060] 2.2. Culture of primary ear tip fibroblasts (PEF) in rats

[0061] Take rat ears, wash with...

Embodiment 3

[0065] Embodiment 3, virus packaging

[0066] 3.1. Amplification of packaging plasmid

[0067] Six kinds of correctly identified vectors obtained in Example 1 were transformed into competent bacteria to amplify, and AxygenAxyPrep TM After pumping in the plasma Maxiprep Kit kit (Axygen Company), purify in an ultra-clean workbench, that is, mix with 1 / 10 volume of 3M NaAC and 2 times the volume of absolute ethanol of the plasmid after pumping, and then centrifuge at 13000rpm After 15 minutes, the supernatant was removed, rinsed with 75% ethanol, sucked off the supernatant, and dried in an ultra-clean workbench. The plasmid was dissolved in sterile deionized distilled water, and finally the concentration of the plasmid was determined using a spectrophotometer and gel electrophoresis.

[0068] 3.2. Transfection

[0069] According to Invitrogen transfection kit (ViraPower TM Lentiviral Expression Systems), using the transfection reagent Lipofectamine TM In 2000, the six le...

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Abstract

The invention relates to rat induced multipotent stem cells and a preparation method thereof. The preparation method for the rat induced multipotent stem cells comprises the following steps: A) constructing a lentiviral vector carrying a transcription factor, wherein the transcription factor may be Oct4, Sox2, c-Myc, Klf4, Lin28 or Nanog; and B) infecting rat adult cells by the lentiviral vector obtained in the step A) in a combination way, selecting the infected adult cells of which the shapes are similar to those of embryonic stem cells for cloning and continuous cell culture, and screening the cells which are in accordance with the characteristics of the embryonic stem cells for cloning to obtain rat iPS cells. The rat induced multipotent stem cells and the preparation method thereof contribute to establishing most suitable culturing conditions and a most suitable culturing method for the cell lines of rat ES cells; the rat iPS cells are good vectors for rat gene targeting; and the rat iPS cells contribute to disclosing various gene functions of a rat and complex development events.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an induced pluripotent stem cell (Induced pluripotent stem cell, iPS cell for short) reprogrammed into a rat adult cell similar to an embryonic stem cell and a preparation method thereof. Background technique [0002] Rats are important experimental animals and are widely used in behavioral, cancer, cardiovascular, blood, autoimmune and other diseases, as well as in physiology and pharmacology. Research on rats is important for guiding biomedicine, human health and pathology, etc. aspect is significant. [0003] Embryonic stem cells are derived from blastocysts, which can multiply indefinitely while maintaining pluripotency, that is, a type of progenitor cell that can differentiate into three germ layers. The reverse genetics method based on embryonic stem cell research has a huge role in promoting genetics, developmental biology, and the establishment of gene knoc...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/68C12Q1/42C12Q1/25G01N33/53
Inventor 肖磊廖婧陈思晔崔春陈霁君任江涛
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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