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

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==Osteogenic differentiation media composition test==
== '''Lentiviral production (BBI_E1)''' ==
*'''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):  
<br>
=== Batch #1 (BBI_E1_T1) ===
*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||
| pLenti CAG Cre IRES Puro ||90ug||-
|-
|-
| Serum ||2ml||1%
| pLenti CMV GFP Puro ||-||30ug
|-
|-
| Dexamethasone ||200ul of 0.1mM stock||0.1uM
| psPAX2 (packaging plasmid) ||60ug||20ug
|-
|-
| Ascorbic acid ||1ml of 10mg/ml stock ||50ug/ml
| pMD2.G (packaging plasmid) ||30ug||10ug
|-
|-
| b-GP ||2ml of 1M stock||10mM
| 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
|-
|-
| PenStrep ||2ml||1%
| C ||10||100
|-
|-
| style="background:#f0f0f0;"|Total
| style="background:#f0f0f0;"|200 mL
| style="background:#f0f0f0;"|
|}
|}
**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).
(2)Aspirate original culture media. Add 1ml virus-containing media to each glass-bottom wells.<br>
**7/2 and 7/3: Cell density was still low. Induction should wait for one more day.
(3)Incubate for 24hrs at 37C. Remove virus-containing media and replace it with fresh osteogenic induction media.
**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.
**7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
 
==Seeding density test==
*'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation
*'''Test #1:'''
**Date: 7/5/2012 ~ ongoing work
**Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
**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.
**7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.

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.