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Hoofed mammal inducible multipotential stem cell and preparation method thereof

A technology of pluripotent stem cells and mammals, applied in the field of induced pluripotent stem cells and their preparation, can solve problems such as hindering the establishment of embryonic stem cell lines, and achieve the effect of improving disease resistance

Inactive Publication Date: 2010-10-20
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, humans have not established embryonic stem cell lines of ungulate mammals, and there are few literature reports on ES cells of such animals in terms of their morphology, surface markers, and pluripotency. There are many controversies in this field in the scientific community further hindered the establishment of such animal embryonic stem cell lines

Method used

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  • Hoofed mammal inducible multipotential stem cell and preparation method thereof
  • Hoofed mammal inducible multipotential stem cell and preparation method thereof
  • Hoofed mammal inducible multipotential stem cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 , Construction of lentiviral vector

[0059] 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).

[0060] Table 1

[0061]

[0062] Note: The uppercase letters in the primer sequences are the restriction restriction sites introduced.

[0063] 1.2. PCR amplification

[0064] Using the total human cDNA as a template, PCR amplification was performed using the primers of each gene in Table 1, as follows:

[0065] 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.

[0066] Reacti...

Embodiment 2

[0070] Example 2 , cell culture

[0071] 2.1. Culture of pig and sheep primary bone marrow mesenchymal cells (BMC)

[0072] Take calf and thigh femur and tibia of newborn pig or sheep, soak them in 75% alcohol for 10 minutes, wash them with PBS containing double antibodies (penicillin, streptomycin) three times, cut the two ends of the bones, and wash with 10% fetal bovine serum (FBS) D-MEM medium sterile syringe into the bone marrow into a 50mL 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 tiny clones were seen. About a week later, the cells were passaged at a ratio of 1:3. During passage, the cells were washed twice with PBS, digested with 0.25% trypsin at 37°C for 5 minutes, and then passaged at a ratio of 1:3 to 4.

[0073] 2.2. Culture of pig and shee...

Embodiment 3

[0079] Example 3 , virus packaging

[0080] 3.1. Amplification of packaging plasmid

[0081] Six kinds of correct vectors obtained in Example 1 were transformed into competent bacteria to be amplified, and Axygen AxyPrep TM After pumping in the plasma Maxiprep Kit kit (Axygen Company), purify in an ultra-clean workbench, that is, mix with 1-fold volume of 3M NaAC and 2-fold volume of absolute ethanol of the plasmid after pumping, and then centrifuge at 10,000 rpm for 15 min. Remove the supernatant, rinse with 75% ethanol, suck off the supernatant, dry it in a clean bench, use sterile deionized distilled water to dissolve the plasmid, and finally use a spectrophotometer to determine the concentration of the plasmid.

[0082] 3.2. Transfection

[0083] According to Invitrogen transfection kit (ViraPower TM Lentiviral Expression Systems), using the transfection reagent Lipofectamine TM In 2000, the six lentiviral plasmids in step 3.1 were combined with the packaging plas...

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Abstract

The invention relates to a hoofed mammal inducible multipotential stem cell and a preparation method thereof. The method comprises the following steps of: A) constructing an expression vector carrying a transcription factor, wherein the transcription factor is Oct4, Sox2, c-Myc, Klf4, Lin28 and Nanog; and B) introducing the transcription factor in the step A) into the cells of the hoofed mammal in a combining form; picking clones of which the form is similar to that of the embryonic stem cell for subculturing; and screening the cell clones meeting the characteristic of the embryonic stem cell to obtain the hoofed mammal iPS cell. The method contributes to determining the most proper culturing condition and the most proper culturing method for establishing an ES cell line of pig, sheep, cattle and other hoofed mammals; and experimental models for various genetic diseases of human beings can be established with the hoofed mammal iPS cell line.

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 an ungulate mammalian adult cell similar to a human embryonic stem cell and a preparation method thereof. Background technique [0002] More than 20 years ago, humans established mouse embryonic stem cell lines (Evans & Kanfman. (1981), Nature 292, 154-156), and in the 1990s, primate embryonic stem cell lines were also established (Thomson, et al. (1995), Proc.Natl.Acad.Sci.92, 7844-7848), the rat embryonic stem cell line has experienced nearly 20 years of research, since its pluripotency gene Oct-4 is difficult to sustain expression (Buehr, et al. (2003), Biol.Repro.68, 222-229), ES-like cells injected into blastocysts can only differentiate into extraembryonic tissues (Demers, et al. (2007), Cloning and stem cells 9, 512-522), to the recent establishment of rat ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 肖磊吴昭陈霁君任江涛鲍磊廖婧崔春饶灵均
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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