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

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==Osteogenic differentiation media composition test==
==Lentiviral production==
*'''Purpose:''' To test the effects of osteogenic differentiation media on mESCs
*'''Purpose:''' To generate and concentrate Cre lentivirus
*'''Test #1:'''
*'''Components:'''
**Date: 7/1/2012 ~ ongoing work
**Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control)
**Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
**Packaging plasmids: psPAX2 and pMD2.G
**Osteogenic differentiation media (with 1% serum):  
*'''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;"|'''Volumn'''
| align="center" style="background:#f0f0f0;"|'''Cre lentivirus'''
| align="center" style="background:#f0f0f0;"|'''Final concentration'''
| align="center" style="background:#f0f0f0;"|'''Control'''
|-
| MEM ||193ml||
|-
| Serum ||2ml||1%
|-
|-
| Dexamethasone ||200ul of 0.1mM stock||0.1uM
| pLenti CAG Cre IRES Puro ||90ug||-
|-
|-
| Ascorbic acid ||1ml of 10mg/ml stock ||50ug/ml
| pLenti CMV GFP Puro ||-||30ug
|-
|-
| b-GP ||2ml of 1M stock||10mM
| psPAX2 (packaging plasmid) ||60ug||20ug
|-
|-
| PenStrep ||2ml||1%
| pMD2.G (packaging plasmid) ||30ug||10ug
|-
|-
| style="background:#f0f0f0;"|Total
| style="background:#f0f0f0;"|Transfected 293T cell number
| style="background:#f0f0f0;"|200 mL
| style="background:#f0f0f0;"|27*10^6 cells
| style="background:#f0f0f0;"|
| style="background:#f0f0f0;"|9*10^6 cells
|}
|}
**7/1: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 10 min, and then seeded 1:5 (two wells) and 1:10 (4 wells) onto feeder free system (now become P10).
**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 and 7/3: Cell density was still low. Induction should wait for one more day.
 
**7/4: Cell density became too high. Several colonies were formed. Therefore, the best time point to induce osteogenic differentiation is ‘’1-2 days after seeding’’’ (need further confirmation).
 
**Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation.
==LentiGFP infection==
**7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
*'''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:
 
 


==Seeding density test==
==LentiCre infection==
*'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation
*'''Purpose:''' To confirm the effect of Cre recomninase in mES brainbow cells
*'''Test #1:'''
*'''Test #1:'''
**Date: 7/5/2012 ~ ongoing work
**Date: 7/1/2012 ~ 7/5/2012
**Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
**Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).
**7/5: P10 mES brainbow cells (on feeder) were trypsinized. Cells were then divided into two groups. Group one were seeded 1:2, 1:4, 1:6, 1:8 and 1:12 on gelatin coated glass-well immediately. Group two were let sit for 15 min and then seeded 1:2, 1:4, and 1:6 on gelatin coated glass-well.
**'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro
**7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.
**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: