Tina:Brainbow induction with Cre recombinase (BBI)
Lentiviral production (E1.1)
- 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 (E1.2)
- Purpose: To confirm the efficiency of virus packaging
- Test #1 (E1.2_T1):
- 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 (E1.3)
- Purpose: To confirm the effect of Cre recomninase in mES brainbow cells
- Test #1 (E1.3_T1):
- 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 (E1.3_T2):
- 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 (E1.3_T3):
- 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 (E1.3_T4):
- Date: 7/28/2012 ~ 8/1/2012
- 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.
- Test #5 (E1.3_T5):
- Date: 8/3/2012 ~ ongoing work
- Cells: P13 mES brainbow cells. P12 mESCs were passaged (let sit for 15 min) 1:6 onto two gelatin coated glass-wells.
- Components: Lentiviruses: pLenti CAG Cre IRES Puro
- Perform osteogenic induction on 8/4:
(1)PBS wash twice.
(2)Change media into osteogenic induction media (with 5% serum).
(3)Change media daily. Incubate for two days.
- Infection on 8/6:
(1)Change MEF-conditioned 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 30 ul LentiCre virus into one glass well.
(4)Put cells back into incubator