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miRNA Detection

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L7ae Repression

Attempt #1

GOAL: Test the strength of L7ae

3

complete

7/14/14: Seed cells

22

complete

7/15/14: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), Mutant hEF1a-2xKturn-eGFP (551.4 ng/uL) and Wild Type hEF1a-2xKturn-eGFP (287.0 ng/uL) into HEK cells

0

complete

7/17/14 (12 pm): Flow Cytometer

DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, 10 uL Mutant Hef1a-2xKturn-eGFP, 20 uL Wild Type Hef1a-2xKturn-eGFP

24-WELL PLATE TRANSFECTION MAP:

(B)1

MAV1212-hEF1a-eBFP2

(B)₂

MAV1212-hEF1a-eBFP2

//////

(G_M)1

Mutant Hef1a-2xKturn-eGFP

(G_M)₂

Mutant Hef1a-2xKturn-eGFP

(G_M+ B)₁

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_M+ B)₂

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_M+ B + L)₁

Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_M+ B + L)₂

Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT)1

Wild Type Hef1a-2xKturn-eGFP

(G_WT)₂

Wild Type Hef1a-2xKturn-eGFP

(G_WT+ B)₁

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT+ B)₂

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT+ B + L)₁

Wild Type Hef1a-2xKturn-eGFP
Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT+ B + L)₂

Wild Type Hef1a-2xKturn-eGFP
Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

Neg Ctrl

Dummy DNA
With Lipofectamine

Neg Ctrl

Dummy DNA
With Lipofectamine

Neg/Neg Ctrl

No DNA
No Lipofectamin

Neg/Neg Ctrl

No DNA
No Lipofectamine

//////

RESULT: Inconclusive. No L7ae was added.

Attempt #2

2

complete

7/16/14: Seed cells

1

complete

7/17/14: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), Mutant hEF1a-2xKturn-eGFP (551.4 ng/uL) and Wild Type hEF1a-2xKturn-eGFP (287.0 ng/uL), MAV1212-hEF1a-L7ae (1686.4 ng/uL) into HEK cells

7

complete

7/19/14 (10 am): Flow Cytometer

DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, 10 uL Mutant Hef1a-2xKturn-eGFP, 20 uL Wild Type Hef1a-2xKturn-eGFP, 3 uL MAV1212-hEF1a-L7ae

24-WELL PLATE TRANSFECTION MAP:

(B)1

MAV1212-hEF1a-eBFP2

(B)₂

MAV1212-hEF1a-eBFP2

//////

(G_M)1

Mutant Hef1a-2xKturn-eGFP

(G_M)₂

Mutant Hef1a-2xKturn-eGFP

(G_M+ B)₁

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_M+ B)₂

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_M+ B + L)₁

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2
MAV1212-hEF1a-L7ae

(G_M+ B + L)₂

Mutant Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2
MAV1212-hEFa-L7ae

(G_WT)1

Wild Type Hef1a-2xKturn-eGFP

(G_WT)₂

Wild Type Hef1a-2xKturn-eGFP

(G_WT+ B)₁

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT+ B)₂

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2

(G_WT+ B + L)₁

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2
MAV1212-hEFa1-L7ae

(G_WT+ B + L)₂

Wild Type Hef1a-2xKturn-eGFP
MAV1212-hEF1a-eBFP2
MAV1212-hEF1a-L7ae

Neg Ctrl

Dummy DNA
With Lipofectamine

Neg Ctrl

Dummy DNA
With Lipofectamine

Neg/Neg Ctrl

No DNA
No Lipofectamin

Neg/Neg Ctrl

No DNA
No Lipofectamine

//////

RESULT: Inconclusive: Too much DNA in each well.

Attempt #3

17

complete

7/24/14: Seed cells

18

complete

7/25/14: Transfect MAV1212-hEF1a-eBFP2, Mutant hEF1a-2xKturn-eGFP, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-L7ae, hEF1a-PirB (dummy DNA) all at ~500 ng/uL into HEK cells

19

complete

7/27/14 (10 am): Flow Cytometry

DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), Wild Type CAG-2xKturn-eGFP (0.96 uL), Mutant CAG-2xKturn-eGFP (0.96 uL), MAV1212-hEF1a-L7ae (0.71 uL), hEF1a-PirB (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1. (B)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

hEF1a-PirB (1.5 fmole = 1.60 uL)

2. (B)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

hEF1a-PirB (1.5 fmole = 1.60 uL)

//////

3. (G_WT+ B)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

4. (G_WT+ B)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

5. (G_WT+ B + L)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

6. (G_WT+ B + L)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

//////

7. (G_M+ B)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

8. (G_M+ B)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

9. (G_M+ B + L)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

10. (G_M+ B + L)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

//////

11. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine

12. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine

13. Neg/Neg Ctrl

No DNA

No Lipofectamine

14. Neg/Neg Ctrl

No DNA

No Lipofectamine

//////

RESULT: Transfection efficiency ___%. Data is available

L7ae Tuning

GOAL: Test the sensitivity of L7ae repression

21

complete

Day One: Seed cells

28

complete

Day Two: Transfect MAV1212-hEF1a-eBFP2, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-L7ae, pEXPR: hEF1a-rtTA (169.0 ng/uL), GG-Donor (dummy DNA) all at ~500 ng/uL into HEK cells

29

complete

Day Three: DOX Induction

30

complete

Day Four (___ am): Flow Cytometry

DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), Wild Type CAG-2xKturn-eGFP (0.96 uL), MAV1212-TRE-L7ae (0.89 uL), pEXPR: hEF1a-rtTA (0.27 uL), GG-Donor (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (1.75 fmole = 1.62 uL)

2.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (1.75 fmole = 1.62 uL)

3.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

Dummy DNA (0.75 fmole = 0.70 uL)

4.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

Dummy DNA (0.75 fmole = 0.70 uL)

//////

5.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

0 uM DOX

6.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

1X DOX

7.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

10X DOX

8.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

100X DOX

9.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

1000X DOX

10.

pEXPR: hEF1a-rtTA

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-TRE-L7ae (1.5 fmoles)

2000X DOX

//////

11.

Dummy DNA (1 ug = ___ uL)

With Lipofectamine

12.

Dummy DNA (1 ug = ___ uL)

With Lipofectamine

13.

No DNA

No Lipofectamine

14.

No DNA

No Lipofectamine

//////

RESULT:Transfection efficiency ___%. Data can be found .

L7ae Quantification

GOAL: Use TASBE tools to quantify the repression of L7ae
32 complete 8/7/14: Seed cells 9 complete 8/8/14: Transfect hEF1a-rtTA (200.0 ng/uL), TRE-L7ae (200 ng/uL), Wild Type 2x-Kturn eGFP (200 ng/uL), TRE-mKate (200.0 ng/ uL), hEF1a-eBFP (200.0 ng/uL), hEF1a-mKate (200.0 ng/uL), hEF1a-eYFP (200.0 ng/uL), and GG Donor (200 ng/uL) into HEK cells 31 complete 8/10/14: (2 pm): Flow Cytometer

DNA Dilution: hEF1a-rtTA (0.60 uL), TRE-L7ae (1.48 uL), Wild Type 2x-Kturn eGFP (1.19 uL), TRE-mKate (1.0 uL), hEF1a-eBFP (0.93 uL), hEF1a-mKate (0.93 uL), hEF1a-eYFP (0.93 uL)

24-WELL PLATE TRANSFECTION MAP:
1. Neg Ctrl
Dummy DNA
With Lipofectamine

2. Neg Ctrl
Dummy DNA
With Lipofectamine

3. Neg/Neg Ctrl
No DNA
No Lipofectamine
4. Neg/Neg Ctrl
No DNA
No Lipofectamine

5.
pEXPR: hEF1a-eYFP (1.0 fmoles)
Dummy DNA (1.5 fmoles = 4.4 uL)

6.
pEXPR: hEF1a-mKate (1.0 fmoles)
Dummy DNA (1.5 fmoles = 4.4 uL)
7.
pEXPR: hEF1a-eBFP (1.0 fmoles)
Dummy DNA (1.5 fmoles = 4.4 uL)
8.
pEXPR: hEF1a-eYFP (1.0 fmoles)
pEXPR: hEF1a-mKate (1.0 fmoles)
pEXPR: hEF1a-eBFP (1.0 fmoles)
Dummy DNA (0.75 fmoles = 2.2 uL)
9.
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
Dummy DNA (1.5 fmoles = 2.4 uL)

10.
pEXPR: hEF1a-rtTA (1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
0 uM DOX
11.
pEXPR: hEF1a-rtTA (1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
1X DOX
12.
pEXPR: hEF1a-rtTA 1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
10X DOX
13.
pEXPR: hEF1a-rtTA (1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
100X DOX
14.
pEXPR: hEF1a-rtTA (1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
1000X DOX
15.
pEXPR: hEF1a-rtTA (1.0 fmoles)
hEF1a-eBFP2 (0.5 fmole)
Wild Type CAG-2xKturn-eGFP (1.0 fmoles)
MAV1212-TRE-L7ae (1.0 fmoles)
pEXPR: TRE-mKate (1.0 fmoles)
2000X DOX

RESULT: Transfection efficiency ___%. Data can be found
L7ae Sensors (without siRNA)
GOAL: Test the sensors in absence of siRNA
10 complete Day 18.11.14: Seed cells 11 incomplete Day 2 complete 8.12.14: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug 12 incomplete Day 4 complete 8.14.14: (___ 4 pm): Flow Cytometer

DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.19 uL), Low Sensor miR-144 (1.29 uL),Low Sensor miR-181c (1.29 uL), High Sensor miR-30d (1.37 uL), High Sensor miR-146a (1.37 uL), High Sensor 125b (1.37 uL), High Sensor let-7f (1.37 uL)

24-WELL PLATE TRANSFECTION MAP:

1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
Dummy DNA (2.9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
Dummy DNA (2.2 uL = 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
Dummy DNA (2.2 uL = 0.75 fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
Dummy DNA (2.2 uL = 0.75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
Dummy DNA (2.2 uL = 0.75 fmoles)
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
Dummy DNA (2.9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
Dummy DNA (2.2 uL = 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
Dummy DNA (2.2 uL = 0.75 fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
Dummy DNA (2.2 uL = 0.75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f (
Dummy DNA (2.2 uL = 0.75 fmoles)
//////
//////
////// ////// ////// //////
13.
pEXPR: hEF1a-mKate
Dummy DNA (5.1uL = 1.75 fmoles)
13.
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP (1.5 fmoles)
Dummy DNA (3.6 uL = 1.25 fmoles)
19.
Dummy DNA
With Lipofectamine
20.
Dummy DNA
With Lipofectamine
21.
No DNA
No Lipofectamine
22.
No DNA
No Lipofectamine

RESULT: Transfection efficiency ___%. Data can be found .
L7ae Sensors (with siRNA)
GOAL: Test the sensors in the presence of siRNA
-1 incomplete 33 complete Day 18.12.14: Seed cells 34 incomplete Day 2 complete 8.13.14: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 35 incomplete Day 4 complete 8.15.14: (___ 4 pm): Flow Cytometer
DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.19 uL), Low Sensor miR-144 (1.29 uL),Low Sensor miR-181c (1.29 uL), High Sensor miR-30d (1.37 uL), High Sensor miR-146a (1.37 uL), High Sensor 125b (1.37 uL), High Sensor let-7f (1.37 uL), 5 uL Mature 144, 5 uL Mature 181c, 5 uL Mature 30d, 5 uL Mature 146a, 5 uL Mature 125b and 5 uL Mature let-7f

24-WELL PLATE TRANSFECTION MAP:
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
siRNA-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
siRNA-181c
Dummy DNA (2.9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
siRNA-30d
Dummy DNA (2.2 uL = 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
siRNA-146a
Dummy DNA (2.2 uL = 0.75 fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
siRNA-125b
Dummy DNA (2.2 uL = 0.75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
siRNA-7f
Dummy DNA (2.2 uL = 0.75 fmoles)
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
siRNA-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
siRNA-181c
Dummy DNA (2.9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
siRNA-30d
Dummy DNA (2.2 uL = 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
siRNA-46a
Dummy DNA (2.2 uL = 0.75 fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
siRNA-125b
Dummy DNA (2.2 uL = 0.75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
siRNA-7f
Dummy DNA (2.2 uL = 0.75 fmoles)
//////
//////
////// ////// ////// //////
13.
pEXPR: hEF1a-mKate
Dummy DNA (5.1uL = 1.75 fmoles)
13.
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP (1.5 fmoles)
Dummy DNA (3.6 uL = 1.25 fmoles)
19.
Dummy DNA
With Lipofectamine
20.
Dummy DNA
With Lipofectamine
21.
No DNA
No Lipofectamine
22.
No DNA
No Lipofectamine

RESULT: Transfection efficiency ___%. Data can be found here .
TAL14 RepressionL7ae Sensors (with and without siRNA)
GOAL: Test the strength of TAL14the sensors in the presence and absence of siRNA
-1 incomplete 14 incomplete Day 1 14 complete 7/25/147/26/14: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 : Seed cells15 complete incompleteDay 2: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells 16 complete 7/28/14 (4 in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 16 incomplete Day 4: (___ pm): Flow Cytometry Cytometer
DNA Dilution: MAV1212-hEF1a-eBFP2 mKate (0.47 46 uL), TAL14Wild Type Kturn-mKate eGFP (01.62 60 uL), hEF1aLow Sensor miR-GAL4VP16 144 (01.72 29 uL), hEF1aLow Sensor miR-TAL14 (___ 181c (1.39 uL), Dummy DNA (to 1 ug)
24-WELL PLATE TRANSFECTION MAP:
High Sensor miR-30d (1.01 uL), High Sensor miR-146a (1.16 uL), High Sensor 125b (1.47 uL), High Sensor let-7f (1.37 uL), 5 uL Mature 144, 5 uL Mature 181c, 5 uL Mature 30d, 5 uL Mature 146a, 5 uL Mature 125b and 5 uL Mature let-7f

24-WELL PLATE TRANSFECTION MAP:
2. (B)₂
MAV1212-hEF1a-eBFP2 (1.0 fmole)5 fmoles = 0.76 uL)3. Neg Ctrl3 fmoles 205 uL
With Lipofectamine
(B + R)₁
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)0 fmoles 61 uL (B + R)₂
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)0 fmoles 61 uL)6. Neg Ctrl3 fmoles 205 uL
With Lipofectamine
//////
10. (B + R + GAL)₁
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 hEF1a-TAL14 (5 12. Neg/Neg Ctrl
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
1. (B)₁
MAV1212-hEF1a-eBFP2 (1.0 fmole)
Dummy DNA (2.5 fmoles = 0.76 uL)
Dummy DNA (2.
////// //////
//////

9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
Dummy DNA (
2.2
uL =
0.
75 fmoles)
4.
miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
Dummy DNA (2.
2 uL = 0.
75 fmoles)
5.
miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
Dummy DNA (2.
2 uL = 0.
//////
////// //////
75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
Dummy DNA (
2.2
uL =
0.
75 fmoles)
7. (B + R + GAL)₁
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.5 fmoles)
8. (B + R + GAL)₂
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.5 fmoles)
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
siRNA-144
Dummy DNA (2.9 uL = 1 fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
siRNA-181c
Dummy DNA (2.9 uL = 1 fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
siRNA-30d
Dummy DNA (2.2 uL = 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
siRNA-146a
Dummy DNA (2.2 uL = 0.75 fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
siRNA-125b
Dummy DNA (2.2 uL = 0.75 fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
siRNA-7f
Dummy DNA (2.2 uL = 0.75 fmoles)
//////
//////
////// ////// ////// //////
13.
pEXPR: hEF1a-mKate
Dummy DNA (5.1uL = 1.75 fmoles)
13.
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
9. Neg/Neg Ctrl
No DNA
No Lipofectamine
(1.5 fmoles)
Dummy DNA (3.6 uL = 1.
25 fmoles)
11. (B + R + GAL)₂
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.5 fmoles)
hEF1a-TAL14 (1.5 fmoles)
//////
////// //////
19.
Dummy DNA
With Lipofectamine
20.
Dummy DNA
With Lipofectamine
21.
No DNA
No Lipofectamine
22.
No DNA
No Lipofectamine

Results: Transfection RESULT: Transfection efficiency ___%. Data can be found .

TAL14 TuningL7ae Sensors (Multi Input)
GOAL: Test the sensors in the presence and absence of siRNA
-1 incomplete 20 complete 9.23.14
GOAL: Determine how much TAL14 is needed to repress TAL14-UAS-mKate
26 complete Day OneDay Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 : Seed cells 27 complete 42 complete 9.24.14: Transfect 1 fmole of Low Sensor miR-144, High Sensor miR-30d, 0.5 fmoles pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells 25 complete Day Four (12:30 in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 43 complete 9.26.14: (___ pm): Flow Cytometry
DNA Dilution: MAV1212-hEF1a-eBFP2 mKate (0.47 46 uL), TAL14Wild Type Kturn-mKate eGFP (01.62 19 uL), hEF1aLow Sensor miR-GAL4VP16 144 (01.72 29 uL), hEF1a-TAL14 (0.22 uL = 1 fmol), Dummy DNA (to 1 ug)High Sensor miR-30d, 5 uL Mature 144, 5 uL Mature 30d

24-WELL PLATE TRANSFECTION MAP:

MAV1212-eBFP2 (1.0 fmole)UAS-TAL14-mKate (1.5 fmoles)1.0 fmoles = 32
1.
Untransfected

2.
pEXPR: hEF1a-mKate
Low Sensor miR-144c MAV1212-hEF1a-eBFP2 (1.0 fmole)
Dummy DNA (2.9 uL = 1 .75 fmoles = 4.06 uL)

//////

//////

//////

//////
3.
pEXPR: hEF1a-mKate
Low Sensor miR-144
2.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
Dummy DNA (2.9 uL = 1 .75 fmoles = 04.06 uL)3
4.
pEXPR: hEF1a-
mKate
High Sensor miR-30d
Dummy DNA (
2.
9 uL = 1 fmoles)5
45.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
Dummy DNA (1.0 fmoles = 2.32 uL)
5.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.5 fmoles)
Dummy DNA (0.75 fmoles = 1.74 uL)
6.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.5 fmoles)
Dummy DNA (0.75 fmoles = 1.74 uL)
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
6.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-30d
7.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-144
8.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-30d
siRNA-144
//////
9.
pEXPR: hEF1a-mKate
High Sensor miR-125b
Dummy DNA (2.9 uL = 1 fmoles)5
10.
pEXPR: hEF1a-mKate
Low Sensor miR-125b
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
11.
pEXPR: hEF1a-mKate
Low Sensor miR-125b
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-30d
12.
pEXPR: hEF1a-mKate
Low Sensor miR-125b
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-125b
13.
pEXPR: hEF1a-mKate
Low Sensor miR-125b
High Sensor miR-30d
Wild Type CAG-2xKturn-eGFP
siRNA-30d
siRNA-125b
//////
14.
pEXPR: hEF1a-mKate
High Sensor miR-181c
Dummy DNA (2.9 uL = 1 fmoles)5
15.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-181c
Wild Type CAG-2xKturn-eGFP
16.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-181c
Wild Type CAG-2xKturn-eGFP
siRNA-181c
17.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-181c
Wild Type CAG-2xKturn-eGFP
siRNA-144
18.
pEXPR: hEF1a-mKate
Low Sensor miR-144
High Sensor miR-181c
Wild Type CAG-2xKturn-eGFP
siRNA-181c
siRNA-144

RESULT: Transfection efficiency ___%. Data can be found .
TAL14 Repression
GOAL: Test the strength of TAL14
44 complete 7/25/14: Seed cells 45 complete 7/26/14: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells 46 complete 7/28/14 (4 pm): Flow Cytometry

DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate (0.62 uL), hEF1a-GAL4VP16 (0.72 uL), hEF1a-TAL14 (___ uL), Dummy DNA (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:
18. UAS-TAL14-mKate (1Gal4VP16
hEF1a-TAL14 (20.0 fmoles)
1. (B)₁
7.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (1.0 fmoles)
8.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (1.5 fmoles)
9.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (2.0 fmoles)
10.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (2.5 fmoles)
11.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (3.0 fmoles)
12.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (3.5 fmoles)
13.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (4.0 fmoles)
14.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (4.5 fmoles)
15.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (5.0 fmoles)
16.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (10.0 fmoles)
17.
MAV1212-hEF1a-eBFP2 (1.0 fmole)
UAS-TAL14-mKate (1.5 fmoles)
hEF1a-Gal4VP16 (1.0 fmoles)
hEF1a-TAL14 (15.0 fmoles)
MAV1212-hEF1a-eBFP2 (1.0 fmole)
Dummy DNA (2.5 fmoles = 0.76 uL)
2. (B)₂
MAV1212-hEF1a-
eBFP2 (1.0 fmole)
Dummy DNA (2.5 fmoles = 0.76 uL)
19.
Dummy DNA (3.2 fmoles = 2.05 uL)
With Lipofectamine
20.
Dummy DNA (3.2 fmoles = 2.05 uL)
With Lipofectamine
21.
No DNA
No Lipofectamine
22.
No DNA
No Lipofectamine

RESULT: Transfection efficiency ___%. Data can be found here.

Toggle Cloak

Treatment

//////

3. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

4. (B + R)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (2.0 fmoles = 0.61 uL)

5. (B + R)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (2.0 fmoles = 0.61 uL)

//////

6. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

7. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

8. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

//////

9. Neg/Neg Ctrl

No DNA

No Lipofectamine

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

hEF1a-TAL14 (1.5 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

hEF1a-TAL14 (1.5 fmoles)

//////

12. Neg/Neg Ctrl

No DNA

No Lipofectamine

Results: Transfection efficiency ___%. Data can be found

TAL14 Tuning

GOAL: Determine how much TAL14 is needed to repress TAL14-UAS-mKate

26

complete

Day One: Seed cells

27

complete

Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells

25

complete

Day Four (12:30 pm): Flow Cytometry

DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate (0.62 uL), hEF1a-GAL4VP16 (0.72 uL), hEF1a-TAL14 (0.22 uL = 1 fmol), Dummy DNA (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (1.75 fmoles = 4.06 uL)

2.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (1.75 fmoles = 04.06 uL)

3.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (1.0 fmoles = 2.32 uL)

4.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (1.0 fmoles = 2.32 uL)

5.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

Dummy DNA (0.75 fmoles = 1.74 uL)

6.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

Dummy DNA (0.75 fmoles = 1.74 uL)

7.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (1.0 fmoles)

8.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (1.5 fmoles)

9.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (2.0 fmoles)

10.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (2.5 fmoles)

11.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (3.0 fmoles)

12.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (3.5 fmoles)

13.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (4.0 fmoles)

14.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (4.5 fmoles)

15.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (5.0 fmoles)

16.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (10.0 fmoles)

17.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (15.0 fmoles)

18.

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (20.0 fmoles)

19.

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

20.

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

21.

No DNA

No Lipofectamine

22.

No DNA

No Lipofectamine

RESULT: Transfection efficiency ___%. Data can be found here.

Toggle Cloak

Treatment

Cloak

Testing effectiveness of miRNA1–miRNA4 in down-regulating BACE1 expression v1

A1 Hef1a: eBFP *250*ng Dummy DNA 750ng Positive Control	A2 Hef1a: eBFP *250ng Hef1a: BACE1 250ng Dummy DNA 500ng* Negative Control	A3 Hef1a: eBFP *250ng TRE: miRNA1-mKATE250ng Hef1a: rtTA 250*ng Dummy DNA 250ng 0 nM Dox	A4 Hef1a: eBFP 250ng TRE: miRNA1-mKATE 250ng Hef1a: rtTA 250ng Hef1a: BACE1 250ng 0 nM Dox	A5 Hef1a: eBFP *250ng* TRE: miRNA1-mKATE *250ng Hef1a: rtTA 250ng Dummy DNA 250ng* 1000 nM Dox	A6 Hef1a: eBFP 250ng TRE: miRNA1-mKATE 250ng Hef1a: rtTA 250ng Hef1a: BACE1 250ng 1000 nM Dox
A7 Hef1a:mKATE 250ng Hef1a:BFP 250ng Dummy DNA 500ng

Testing inducible expression of BACE2

B1 Hef1a:eBFP 333ng Dummy DNA 666ng Negative Control	B2 Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 0 nM Dox	B3 Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 1000 nM Dox	B4 No DNA (untransfected) Double negative control

Transfection: Week of 8/11

A1

- Hef1a: eBFP 250ng
- Dummy DNA 750ng

Negative BACE1 Control

A2

- Hef1a: eBFP 250ng
- Hef1a: BACE1 250ng
- Dummy DNA 500ng

Positive BACE1 Control

A3

- Hef1a:mKATE 250ng
- Hef1a:BFP 250ng
- Dummy DNA 500ng

mKATE control

A4

- Hef1:miRNAg1 250ng
- Hef1a:BFP 250ng
- Dummy DNA 500ng

miRNA1g control

A5

- Hef1a: eBFP 250ng
- TRE: miRNA1-mKATE250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  
  0 nM Dox

A6

- Hef1a: eBFP 250ng
- TRE: miRNA1-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 nM Dox

A7

- Hef1a: eBFP 250ng

Cloak

Testing effectiveness of miRNA1–miRNA4 in down-regulating BACE1 expression v1

A1

Hef1a: eBFP 250ng

Dummy DNA 750ng
Positive ControlA7mKATE

Hef1a:BFP 250ng

Dummy DNA 500ng

A2

- Hef1a: eBFP 250ng
- Hef1a: BACE1 250ng
- Dummy DNA 500ng

Negative Control

A3

- Hef1a: eBFP 250ng
- TRE: miRNA1-mKATE 250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  0
  1000 nM Dox

A4A8

- Hef1a: eBFP 250ng
- TRE: miRNA1-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 1000 nM Dox

A5A9

- Hef1a: eBFP 250ng 250ng
- TRE: miRNA1miRNA2-mKATE250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  1000
  0 nM Dox

A6A10

- Hef1a: eBFP 250ng
- TRE: miRNA1miRNA2-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000 0 nM Dox

A11

- Hef1a:

- eBFP 250ng

Testing inducible expression of BACE2

- TRE: miRNA2-mKATE 250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  
  1000 nM Dox

A12

- Hef1a: eBFP 250ng
- TRE: miRNA2-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

B1 Hef1a:eBFP 333ng Dummy DNA 666ng Negative Control	B2 Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 0 nM Dox	B3 Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 1000 nM Dox	B4 No DNA (untransfected) Double negative control

Transfection: Week of 8/11

A13

- Hef1a: eBFP 250ng
- TRE: miRNA3-mKATE250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  
  0 nM Dox

A14

- Hef1a: eBFP 250ng
- TRE: miRNA3-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 nM Dox

A15

- Hef1a: eBFP 250ng
- TRE: miRNA3-mKATE

A1

Hef1a: eBFP 250ng

Dummy DNA 750ng

Negative BACE1 Control

A2

- Hef1a: eBFP 250ng
- Hef1a: BACE1 rtTA 250ng
- Dummy DNA 500ng 250ng

Positive BACE1 Control

- 1000 nM Dox

A16 A3

- Hef1a: mKATE eBFP 250ngHef1a
- TRE: BFP miRNA3-mKATE 250ng
- Dummy DNA 500ng

mKATE control

A4

- Hef1:miRNAg1 Hef1a: rtTA 250ng
- Hef1a: BFP BACE1 250ng
- Dummy DNA 500ng

miRNA1g control

1000 nM Dox

A17A5

- Hef1a: eBFP 250ng
- TRE: miRNA1miRNA4-mKATE250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  
  0 nM Dox

A6A18

- Hef1a: eBFP 250ng
- TRE: miRNA1miRNA4-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 nM Dox

A7A19

- Hef1a: eBFP 250ng
- TRE: miRNA1miRNA4-mKATE 250ng
- Hef1a: rtTA 250ng
- Dummy DNA 250ng
  
  1000 nM Dox

A8A20

- Hef1a: eBFP 250ng
- TRE: miRNA1miRNA4-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000 nM Dox

B1

- Hef1a:eBFP 333ng
- Dummy DNA 666ng

Negative Control

B2

- Hef1a:eBFP 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

0 nM Dox

A9B3

- Hef1a:eBFP 250ng 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

1000 nM Dox

B4

- No DNA (untransfected)

Double negative control

Transfection 9/17

A1

- TRE: miRNA2-mKATE250ng
- Hef1a: eBFP
- 250ng
- Dummy DNA

A10

- Hef1a: eBFP 250ng
- TRE: miRNA2-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 nM Dox

- 750ng

Negative BACE1 Control

A2

- Hef1a: eBFP

A11

- Hef1a: eBFP 250ng
- TRE: miRNA2-mKATE 250ng
- Hef1a: rtTA BACE1 250ng
- Dummy DNA 250ng
  1000 nM Dox 500ng

Positive BACE1 Control

A3A12

- Hef1a: eBFP 250ngTRE: miRNA2-mKATE 250ng
- Hef1a: rtTA BFP 250ng
- Hef1a: BACE1 250ng
- Dummy DNA 500ng

mKATE control

A4

1000 nM Dox

A13

- Hef1a: eBFP 250ng 250ng
- TRE: miRNA3miRNA1-mKATE 250ng 250ng
- Hef1a: rtTA 250ng 250ng
- Hef1a: BACE1 Dummy DNA250ng

0 nM Dox

A5A14

- Hef1a: eBFP 250ng
- TRE: miRNA3miRNA1-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 1000 nM Dox

A6A15

- Hef1a: eBFP 250ng
- TRE: miRNA3miRNA2-mKATE 250ng 250ng
- Hef1a: rtTA 250ng 250ng
- Hef1a: BACE1 Dummy DNA 250ng1000

0 nM Dox

A16A7

- Hef1a: eBFP 250ng
- TRE: miRNA3miRNA2-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000 nM Dox

A17A8

- Hef1a: eBFP 250ng 250ng
- TRE: miRNA4miRNA3-mKATE 250ng 250ng
- Hef1a: rtTA 250ng 250ng
- Hef1a: BACE1 Dummy DNA250ng

0 nM Dox

A18 A9

- Hef1a: eBFP 250ng
- TRE: miRNA4miRNA3-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 1000 nM Dox

A19A10

- Hef1a: eBFP 250ng
- TRE: miRNA4-mKATE 250ng 250ng
- Hef1a: rtTA 250ng 250ng
- Hef1a: BACE1 Dummy DNA 250ng1000

0 nM Dox

A20A11

- Hef1a: eBFP 250ng
- TRE: miRNA4-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000 nM Dox

B1

- Hef1a:eBFP 333ng
- Dummy DNA 666ng

Negative Control

B2

- Hef1a:eBFP 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

0 nM Dox

B3

- Hef1a:eBFP 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

1000 nM Dox

B4

- No DNA (untransfected)

Double negative control

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