Tina:Brainbow induction with Cre recombinase (BBI): Difference between revisions
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== | ==Lentiviral production== | ||
*'''Purpose:''' To | *'''Purpose:''' To generate and concentrate Cre lentivirus | ||
*''' | *'''Components:''' | ||
**Date: | **Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control) | ||
**Cells: | **Packaging plasmids: psPAX2 and pMD2.G | ||
** | *'''Batch #1:''' | ||
**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'''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Cre lentivirus''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Control''' | ||
|- | |- | ||
| | | pLenti CAG Cre IRES Puro ||90ug||- | ||
|- | |- | ||
| | | pLenti CMV GFP Puro ||-||30ug | ||
|- | |- | ||
| | | psPAX2 (packaging plasmid) ||60ug||20ug | ||
|- | |- | ||
| | | pMD2.G (packaging plasmid) ||30ug||10ug | ||
|- | |- | ||
| style="background:#f0f0f0;"| | | style="background:#f0f0f0;"|Transfected 293T cell number | ||
| style="background:#f0f0f0;"| | | style="background:#f0f0f0;"|27*10^6 cells | ||
| style="background:#f0f0f0;"| | | 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. | ||
**7/2 | |||
**7/ | |||
==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 | *'''Purpose:''' To confirm the effect of Cre recomninase in mES brainbow cells | ||
*'''Test #1:''' | *'''Test #1:''' | ||
**Date: 7/5/2012 | **Date: 7/1/2012 ~ 7/5/2012 | ||
**Cells: | **Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells). | ||
**7/ | **'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro | ||
**7/ | **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: |
Revision as of 18:10, 23 July 2012
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: