Tina:Brainbow induction with Cre recombinase (BBI)

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Revision as of 02:10, 31 July 2012 by >TinaLo (→‎LentiCre infection)
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Lentiviral production

  • Purpose: To generate and concentrate Cre lentivirus
  • Components:
    • Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control)
    • Packaging plasmids: psPAX2 and pMD2.G
  • Batch #1:
    • Date: 6/26/2012 ~ 6/29/2012
    • Cells: 293T cells
    • Virus packaging components:
Component'' Cre lentivirus Control
pLenti CAG Cre IRES Puro 90ug -
pLenti CMV GFP Puro - 30ug
psPAX2 (packaging plasmid) 60ug 20ug
pMD2.G (packaging plasmid) 30ug 10ug
Transfected 293T cell number 27*10^6 cells 9*10^6 cells
    • Virus production: Exp was carried out at Dr. Yang Xu’s Lab (with Dr. Blue Lake’s help). 293T cells were seeded on 6/26 and transfected on 6/27. Virus were collected and concentrated on 6/29. Aliquot (105 ul) virus are stored at -80゚C at CERC.


LentiGFP infection

  • Purpose: To confirm the efficiency of virus packaging
  • Test #1:
    • Date: 7/2/2012 ~ 7/6/2012
    • Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto one gelatin coated glass-well.
    • Components: Lentiviruses: pLenti CMV GFP Puro
    • Infection on 7/3:

(1)Take pictures of cells before viral infection

(2)Change media of each well (2 ml/well)

(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency

(4)Add 50 ul LentiCre virus in culture medium, and then mix gently

(5)Put cells back into incubator

(6)Take pictures of cells 2-3 days after infection.

    • Results:

Observation: Successful infection of GFP vectors. Both mESCs and MEFs are expressing GFPs now. Efficiency is higher in MEFs.

Pictures:


LentiCre infection

  • Purpose: To confirm the effect of Cre recomninase in mES brainbow cells
  • Test #1:
    • Date: 7/1/2012 ~ 7/5/2012
    • Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).
    • Components: Lentiviruses: pLenti CAG Cre IRES Puro
    • Infection on 7/3:

(1)Take pictures of cells before viral infection

(2)Change media of each well (2 ml/well)

(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency

(4)Add 0/10/25/50 ul LentiCre virus in culture medium, and then mix gently

(5)Put cells back into incubator

(6)Take pictures of cells 2-3 days after infection.

    • Results:


  • Test #2:
    • Date: 7/2/2012 ~ 7/6/2012
    • Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto gelatin coated glass-well (total: four wells).
    • Components: Lentiviruses: pLenti CAG Cre IRES Puro
    • Infection on 7/3:

(1)Take pictures of cells before viral infection

(2)Change media of each well (2 ml/well)

(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency

(4)Add 0/10 ul LentiCre virus in culture medium, and then mix gently

(5)Put cells back into incubator

(6)Take pictures of cells 2-3 days after infection.

    • Results:


  • Test #3:
    • Date: 7/16/2012 ~ 7/20/2012
    • Cells: P11 mES brainbow cells. P10 mESCs were passaged (let sit for 15 min) 1:6 onto gelatin coated glass-well (total: two wells).
    • Components: Lentiviruses: pLenti CAG Cre IRES Puro
    • Infection on 7/17:

(1)Change media of each well (2 ml/well)

(2)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency

(3)Add 0/10 ul LentiCre virus in culture medium, and then mix gently

(4)Put cells back into incubator

    • Take pictures of cells 2-3 days after infection.
    • Results:


  • Test #4:
    • Date: 7/28/2012 ~ ongoing work
    • Cells: P11 mES brainbow cells. P10 mESCs were passaged (let sit for 15 min) 1:4 onto four gelatin coated glass-wells and two MEFs co-cultured 35mm dishes.
    • Components: Lentiviruses: pLenti CAG Cre IRES Puro
    • Infection on 7/29:

(1)Change media of each well (2 ml/well)

(2)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency

(3)Add 0/10/25/50 ul LentiCre virus into glass well, respectively, and 0/25 ul LentiCre virus into MEFs co-cultured 35mm dishes.

(4)Put cells back into incubator

    • Take pictures of cells 2-3 days after infection.