Tina:Brainbow induction with Cre recombinase (BBI): Difference between revisions

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==Lentiviral production (E1.1)==
== '''Lentiviral production (BBI_E1)''' ==
*'''Purpose:''' To generate and concentrate Cre lentivirus
*'''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
*'''Batch #1:'''
<br>
**Date: 6/26/2012 ~ 6/29/2012
=== Batch #1 (BBI_E1_T1) ===
**Cells: 293T cells
*Date: 6/26/2012 ~ 6/29/2012
**Virus packaging components:  
*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.
*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)==
== '''LentiGFP infection (BBI_E2)''' ==
*'''Purpose:''' To confirm the efficiency of virus packaging
*'''Purpose: To confirm the efficiency of virus packaging'''
*'''Test #1 (''E1.2_T1''):'''
<br>
**Date: 7/2/2012 ~ 7/6/2012
=== Test #1 (BBI_E2_T1) ===
**Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto one gelatin coated glass-well.
*Date: 7/2/2012 ~ 7/6/2012
**Components: Lentiviruses: pLenti CMV GFP Puro  
*Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto one gelatin coated glass-well.
**Infection on 7/3:
*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:
*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 (E1.3)==
== '''LentiCre infection (BBI_E3)''' ==
*'''Purpose:''' To confirm the effect of Cre recomninase in mES brainbow cells
*'''Purpose: To confirm the effect of Cre recomninase in mES brainbow cells'''
*'''Test #1 (''E1.3_T1''):'''
<br>
**Date: 7/1/2012 ~ 7/5/2012
=== Test #1 (BBI_E3_T1) ===
**Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).  
*Date: 7/1/2012 ~ 7/5/2012
**Components: Lentiviruses: pLenti CAG Cre IRES Puro  
*Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).  
**Infection on 7/3:
*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:
*Results:
   
   




*'''Test #2 (''E1.3_T2''):'''
=== Test #2 (BBI_E3_T2) ===
**Date: 7/2/2012 ~ 7/6/2012
*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).  
*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  
*Components: Lentiviruses: pLenti CAG Cre IRES Puro  
**Infection on 7/3:
*Infection on 7/3: Add 0/10 ul LentiCre virus in culture medium.Take pictures of cells 2-3 days after infection.
(1)Take pictures of cells before viral infection
*Results:
 
(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:
=== Test #3 (BBI_E3_T3) ===
(1)Change MEF-conditioned media of each well (2 ml/well)
*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:


(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
=== 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.