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Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method

A technology of pluripotent stem cells and construction methods, applied in the field of induced pluripotent stem cells, porcine induced pluripotent stem cells and their construction, can solve the problems of increasing the risk of carcinogenesis of iPS cells, and achieve the effect of reducing the risk of carcinogenesis

Inactive Publication Date: 2012-10-24
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

However, exogenous genes (Oct4, Sox2 and Klf4, c-Myc or Nanog, Lin28) are integrated in these porcine iPS cells, and the presence of exogenous genes will increase the cancer risk of these iPS cells. Integrated iPS cells are particularly important

Method used

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  • Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method
  • Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method
  • Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1, construction of lentiviral vector Lv-ef1α-Cre

[0039]1.1. Design Cre primers according to the sequence of pBluescript II SK(-)-Cre (Stratagene), and introduce restriction sites. The primer sequences are shown in Table 1 (wherein F represents the forward primer and R represents the reverse primer).

[0040] Table 1 Primer Sequence

[0041]

[0042] Note: The lowercase letters in the primer sequences are the restriction restriction sites introduced.

[0043] 1.2PCR amplification

[0044] Using the pBluescript II SK(-)-Cre plasmid as a template, use the primers in Table 1 for PCR amplification, as follows:

[0045] Reaction system (25 μl): template 0.3 μl, forward and reverse primers 0.25 μl, 10×pfx Mix 2.5 μl, Accuprime pfx enzyme 0.2 μl, double distilled water 21.5 μl.

[0046] Reaction conditions: 95°C for 2min; 95°C for 15sec, 66°C for 20sec, 68°C for 1min, 35 cycles; 68°C for 10min.

[0047] 1.3. PCR product purification, enzyme digestion, ligation ...

Embodiment 2

[0051] Example 2, construction of lentiviral vector Lv-ef1α-CreER

[0052] 2.1. Design CreER primers according to the sequence of pCAGG-CreER (Stratagene), and introduce restriction sites. The primer sequences are shown in Table 1 (wherein F represents the forward primer and R represents the reverse primer).

[0053] 2.2. PCR amplification

[0054] Using the pCAGG-CreER plasmid as a template, PCR amplification was performed using the primers in Table 1, as follows:

[0055] Reaction system (25ul): template 0.3μl, forward and reverse primers 0.25μl each, 10×pfx Mix 2.5μl, Accuprime pfx enzyme 0.2μl, double distilled water 21.5μl.

[0056] Reaction conditions: 95°C for 2min; 95°C for 15sec, 66°C for 20sec, 68°C for 2min, 35 cycles; 68°C for 10min.

[0057] 2.3. PCR product purification, enzyme digestion, ligation

[0058] The resulting PCR product was subjected to agarose gel electrophoresis to determine its concentration. Use the universal DNA purification and recovery kit ...

Embodiment 3

[0060] Transform the ligation product into GBE180 competent bacteria, spread it evenly on the agar plate containing ampicillin antibiotics, and cultivate it at 37°C for 16 hours, pick an appropriate number (10-20) of single colonies from the agar plate, and put it on the agar plate containing Shake culture in a 15ml round bottom centrifuge tube with 5ml LB medium for 16 hours. Use the Biotech B-type Plasmid Small Quantity Rapid Extraction Kit to extract the plasmid, use BamH I and Sal I double enzyme digestion to identify, select the plasmid with the correct digestion and send it to the sequencing company for sequencing, after sequencing identification, the correct Lv-ef1α-CreER was obtained , with a structure such as figure 2 As shown, 8539bp. Example 3. Mutation of the EcoR I restriction site behind the ER in the lentiviral vector Lv-ef1α-CreER to Nhe I

[0061] 3.1. PCR mutation primer design

[0062] In the Lv-ef1α-CreER sequence, about 20 bp were designed on both side...

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Abstract

The invention relates to a non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method. The method comprises the steps of: A) infecting a porcine induced pluripotent stem cell with a CreER lentiviral vector, inducing the infected stem cell with 4OH-Tamoxifen, cutting off loxp-carrying exogenous factors Oct4, Sox2, Klf4 and c-Myc by CreER; and B) screening out a stem cell that does not express green fluorescent protein by means of FACS, i.e. the non-exogenous induced factor integrated porcine induced pluripotent stem cell. The non-exogenous induced factor integrated porcine induced pluripotent stem cell obtained in the invention can be applied in nuclear transfered cloned pigs or transgenic pigs, thus greatly reducing the carcinogenic risk of exogenous induced factors, especially c-Myc.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to an induced pluripotent stem cell (Induced pluripotent stem cell, iPS cell for short), in particular to a porcine inducible pluripotent stem cell (pig iPS cell) without exogenous inducing factor integration and its build method. Background technique [0002] Embryonic stem cells (ES) are cells obtained from the inner cell mass (ICM) of mammalian blastocysts through in vitro culture, and are characterized by unlimited proliferation and developmental totipotency. [0003] Embryonic stem cells are of great significance to life science research, and their application prospects are very broad: (1) Cell transplantation therapy, ES cells can expand infinitely in vitro and develop into various adult cells, so ES cells are important for cell transplantation therapy Cell sources, such as ES cells differentiated into pancreatic islet cells to treat type 1 diabetes; (2) cell models for study...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 肖磊朱辉陈霁君吴璐
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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