Tina:Brainbow induction with Cre recombinase (BBI): Difference between revisions
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==Lentiviral production ( | == '''Lentiviral production (BBI_E1)''' == | ||
*'''Purpose: | *'''Purpose: To generate and concentrate Cre lentivirus''' | ||
*Components: | *Components: | ||
**Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control) | **Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control) | ||
**Packaging plasmids: psPAX2 and pMD2.G | **Packaging plasmids: psPAX2 and pMD2.G | ||
<br> | |||
=== Batch #1 (BBI_E1_T1) === | |||
*Date: 6/26/2012 ~ 6/29/2012 | |||
*Cells: 293T cells | |||
*Virus packaging components: | |||
{| {{table}} border=1 | {| {{table}} border=1 | ||
| align="center" style="background:#f0f0f0;"|''Component'''' | | align="center" style="background:#f0f0f0;"|''Component'''' | ||
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| style="background:#f0f0f0;"|9*10^6 cells | | style="background:#f0f0f0;"|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 ( | == '''LentiGFP infection (BBI_E2)''' == | ||
*'''Purpose: | *'''Purpose: To confirm the efficiency of virus packaging''' | ||
<br> | |||
=== Test #1 (BBI_E2_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 | (1)Take pictures of cells before viral infection | ||
Line 47: | Line 49: | ||
(6)Take pictures of cells 2-3 days after infection. | (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. | Observation: Successful infection of GFP vectors. Both mESCs and MEFs are expressing GFPs now. Efficiency is higher in MEFs. | ||
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==LentiCre infection ( | == '''LentiCre infection (BBI_E3)''' == | ||
*'''Purpose: | *'''Purpose: To confirm the effect of Cre recomninase in mES brainbow cells''' | ||
<br> | |||
=== Test #1 (BBI_E3_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 | (1)Take pictures of cells before viral infection | ||
Line 73: | Line 76: | ||
(6)Take pictures of cells 2-3 days after infection. | (6)Take pictures of cells 2-3 days after infection. | ||
*Results: | |||
=== Test #2 (BBI_E3_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: Add 0/10 ul LentiCre virus in culture medium.Take pictures of cells 2-3 days after infection. | |||
*Results: | |||
** | === Test #3 (BBI_E3_T3) === | ||
(1 | *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: Add 0/10 ul LentiCre virus in culture medium. Take pictures of cells 2-3 days after infection. | |||
*Results: | |||
( | === Test #4 (BBI_E3_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: Add 0/10/25/50 ul LentiCre virus into glass well, respectively, and 0/25 ul LentiCre virus into MEFs co-cultured 35mm dishes. Take pictures of cells 2-3 days after infection. |
Revision as of 22:12, 8 August 2012
Lentiviral production (BBI_E1)
- 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 (BBI_E1_T1)
- 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 (BBI_E2)
- Purpose: To confirm the efficiency of virus packaging
Test #1 (BBI_E2_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 (BBI_E3)
- Purpose: To confirm the effect of Cre recomninase in mES brainbow cells
Test #1 (BBI_E3_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 (BBI_E3_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: Add 0/10 ul LentiCre virus in culture medium.Take pictures of cells 2-3 days after infection.
- Results:
Test #3 (BBI_E3_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: Add 0/10 ul LentiCre virus in culture medium. Take pictures of cells 2-3 days after infection.
- Results:
Test #4 (BBI_E3_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: Add 0/10/25/50 ul LentiCre virus into glass well, respectively, and 0/25 ul LentiCre virus into MEFs co-cultured 35mm dishes. Take pictures of cells 2-3 days after infection.