Tina:Brainbow induction with Cre recombinase (BBI): Difference between revisions
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*Virus packaging components: | *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;"|'''Cre lentivirus''' | | align="center" style="background:#f0f0f0;"|'''Cre lentivirus''' | ||
| align="center" style="background:#f0f0f0;"|'''Control''' | | align="center" style="background:#f0f0f0;"|'''Control''' | ||
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**Gene: tamoxifen inducible Cre (CreERT2) | **Gene: tamoxifen inducible Cre (CreERT2) | ||
=== Test #1 (BBI_E4_T1) === | === Test #1 (BBI_E4_T1) === | ||
*Date: 4/10/2013 ~ | *Date: 4/10/2013 ~ 4/12/2013 | ||
*Cells: P10 H9Bb3. H9 ESC brainbow clone #3. Feeder free culture system. Cell seeded on 4/10 in three glass bottom wells. | *Cells: P10 H9Bb3. H9 ESC brainbow clone #3. Feeder free culture system. Cell seeded on 4/10 in three glass bottom wells. | ||
*Infection on 4/ | *Results: experiment terminated. Cell type is not suitable for adenovirus infection. Because adenovirus will not integrate into host genome, proliferating cells (ESCs here) are not good starting material for Ad-Cre infection. Most of the cells won't be expressing Cre after 2-3 days. Will try to use differentiated cells (ESC-derived osteoblasts) next time. As long as cells are not proliferating, most of the cells should be able to express Cre. | ||
=== Test #2 (BBI_E4_T2) === | |||
*Date: 4/17/13 ~ | |||
*Cells: P11 H9Bb3 derived osteoblasts. Feeder free culture system. Cell seeded on 4/17 in three glass bottom wells and started osteogenic differentiation (5% serum osteogenic medium) on 4/20. | |||
*Viruss: Adenoviruses: Ad-CreERT2, 10e10 PFU/ml | |||
*Observations: After osteogenic differentiation, cells remain proliferating for a while. In order to increase transfection efficiency, I wait for a few more days until these cells stop dividing (on 4/26, 6 days after induction). | |||
*Infection on 4/26: | |||
(1)Prepare virus-containing media: | |||
{| {{table}} border=1 | |||
| align="center" style="background:#f0f0f0;"|'''Group''' | |||
| align="center" style="background:#f0f0f0;"|'''amount of virus (ul/ 1 ml medium)''' | |||
| align="center" style="background:#f0f0f0;"|'''MOI (multiplicity of infection)''' | |||
|- | |||
| A ||1||10 | |||
|- | |||
| B ||5||50 | |||
|- | |||
| C ||10||100 | |||
|- | |||
|} | |||
(2)Aspirate original culture media. Add 1ml virus-containing media to each glass-bottom wells.<br> | |||
(3)Incubate for 24hrs at 37C. Remove virus-containing media and replace it with fresh osteogenic induction media. |
Latest revision as of 23:05, 26 April 2013
Lentiviral production (BBI_E1)[edit]
- 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)[edit]
- 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)[edit]
- Purpose: To confirm the efficiency of virus packaging
Test #1 (BBI_E2_T1)[edit]
- 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)[edit]
- Purpose: To confirm the effect of Cre recomninase in mES brainbow cells
Test #1 (BBI_E3_T1)[edit]
- 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)[edit]
- 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)[edit]
- 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)[edit]
- 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.
Ad-CreERT2 infection (BBI_E4)[edit]
- Purpose: Use Adenovirus infection to avoid genome integration of Cre recombinase gene
- Components: Ad-CreERT2 from Vector Biolabs (Cat.# 1794)
- Viral backbone: Adenovirus-type 5 (dE1/E3)
- Promoter: CMV
- Gene: tamoxifen inducible Cre (CreERT2)
Test #1 (BBI_E4_T1)[edit]
- Date: 4/10/2013 ~ 4/12/2013
- Cells: P10 H9Bb3. H9 ESC brainbow clone #3. Feeder free culture system. Cell seeded on 4/10 in three glass bottom wells.
- Results: experiment terminated. Cell type is not suitable for adenovirus infection. Because adenovirus will not integrate into host genome, proliferating cells (ESCs here) are not good starting material for Ad-Cre infection. Most of the cells won't be expressing Cre after 2-3 days. Will try to use differentiated cells (ESC-derived osteoblasts) next time. As long as cells are not proliferating, most of the cells should be able to express Cre.
Test #2 (BBI_E4_T2)[edit]
- Date: 4/17/13 ~
- Cells: P11 H9Bb3 derived osteoblasts. Feeder free culture system. Cell seeded on 4/17 in three glass bottom wells and started osteogenic differentiation (5% serum osteogenic medium) on 4/20.
- Viruss: Adenoviruses: Ad-CreERT2, 10e10 PFU/ml
- Observations: After osteogenic differentiation, cells remain proliferating for a while. In order to increase transfection efficiency, I wait for a few more days until these cells stop dividing (on 4/26, 6 days after induction).
- Infection on 4/26:
(1)Prepare virus-containing media:
Group | amount of virus (ul/ 1 ml medium) | MOI (multiplicity of infection) |
A | 1 | 10 |
B | 5 | 50 |
C | 10 | 100 |
(2)Aspirate original culture media. Add 1ml virus-containing media to each glass-bottom wells.
(3)Incubate for 24hrs at 37C. Remove virus-containing media and replace it with fresh osteogenic induction media.