Method for transfecting pig T lymphocytes in vitro by applying nuclear transfection method

Method for transfecting pig T lymphocytes in vitro by applying nuclear transfection method

  • CN 102,206,678 B
  • Filed: 04/12/2011
  • Issued: 07/15/2015
  • Est. Priority Date: 04/12/2011
  • Status: Active Grant
First Claim
Patent Images

1. apply the outer lymphocytic method of By Transfecting Porcine T of consideration convey dye body of laws, comprise the steps:

  • that GFP mRNA imports in pig peripheral blood monocyte (PBMC) by the nucleus transfection liquid applying electroporation technology and cell-specific, cell after transfection is through cultivating, sub-electing the Swine PBMC of expression alien gene, and the concentration of described Swine PBMC in the nucleus transfection liquid of described cell-specific is 3 ×

    10 6/ 100 μ

    l mouse T cell transfection liquid, every 3 ×

    10 6described Swine PBMC adds 10 μ

    g or 20 μ

    gGFP mRNA;

    Wherein, described Swine PBMC is initial Swine PBMC or the Swine PBMC of activation;

    The Swine PBMC of described activation is by 3 ×

    10 6/ ml PBMCs carries out activation with 2 μ

    g/ml concanavalin As and obtains for two days;

    Described core infection protocol uses plasmid-mediated;

    Wherein, the operation steps of described plasmid-mediated core infection protocol is as follows;

    (1) a large amount of extractions of pMaxGFP (AMAXA) plasmid;

    The E.Z.N.A.TM Endo-Free Plasmid Maxi Kit specification sheets produced according to OMEGA company carries out, after plasmid extraction completes, by the concentration of spectrophotometric determination plasmid;

    (2) extraction of pig peripheral blood mononuclearcell (PBMC);

    Take 20ml heparin sodium anticoagulation from the precaval vein of pig is aseptic, after the dilution of PBS equal-volume, mixing, it is slowly added on isopyknic pig lymphocyte parting liquid, the centrifugal 1800rpm of horizontal rotor, 20min, draws the buffy coat under blood plasma afterwards, obtains PBMC;

    (3) transfection of pig peripheral blood PBMC;

    After cell counting, by 5 ×

    10 6pMBC that is unactivated or that activate is dissolved in 100 μ

    l and returns in the mouse lymphotactin transfection damping fluid of room temperature, adds 4 μ

    g pMaxGFP, sets up negative group that does not add plasmid simultaneously, transfer in 2mm consideration convey cup gently after mixing;

    Consideration convey cup is put into consideration convey instrument, transfection T cells is carried out respectively with the intrinsic program X001 in consideration convey instrument and Z001 program, X001 and Z001 program carries out the T cell of transfection activation respectively, then cell is transferred in complete 1640 substratum of 2ml37 DEG C of preheating rapidly, and be placed in 37 DEG C, 5%CO 2cell culture incubator in cultivate;

    The PMBC activated;

    first by 3 ×

    10 6/ ml PBMCs stimulates two days through 2 μ

    g/ml conA;

    (4) detection of T cell transfection efficiency and cell survival rate;

    After transfection 24h, by cell from the careful sucking-off of cell plate, centrifugation, and count;

    Then 1 ×

    10 is got 6cell dyes;

    by 10 6cell is suspended from 100 μ

    l and contains in the dye solution of the PBS of 0.5%BSA and 2mM EDTA, adds the mouse-anti pig CD3 ε

    antibody that 0.2 μ

    g fluorescein PE marks, and mixing is placed on reacts 20 minutes on ice;

    Clean three times with PBS afterwards, wash away non-binding antibody;

    Cell is suspended from 100 μ

    1 dye solution all over after washing by again, adds 0.25 μ

    g 7-AAD, and mixing is placed on reacts 10 minutes on ice;

    Appropriate PBS is directly added, with the surviving rate of flow cytomery cell and the transfection efficiency of T cell after reaction.

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