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Tina:Brainbow induction with Cre recombinase (BBI)
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== '''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 <br> === Batch #1 (BBI_E1_T1) === *Date: 6/26/2012 ~ 6/29/2012 *Cells: 293T cells *Virus packaging components: {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Cre lentivirus''' | 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;"|Transfected 293T cell number | style="background:#f0f0f0;"|27*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 (BBI_E2)''' == *'''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 (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''' <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 (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. == '''Ad-CreERT2 infection (BBI_E4)''' == *'''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) === *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) === *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.
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